Plant Molecular Biology 7:201-205 (1986) © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands
201
Restriction fragment map of sugar beet ( B e t a vulgaris L . ) chloroplast D N A Yuji Kishima, 1 Tetsuo Mikami, 1 Takeo Harada, 1,* Kazuo Shinozaki, 2 Masahiro Sugiura 2 & Toshiro Kinoshital, ** 1Plant Breeding Institute, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan; 2Center for Gene Research, Nagoya University, Chikusa, Nagoya 464, Japan; *Present address: Hokkaido Central Agricultural Experiment Station, Naganuma 069-13, Japan
Keywords: Beta vulgaris, chloroplast DNA, electron microscopy, restriction fragment map, sugar beet
Summary A restriction endonuclease fragment map of sugar beet chloroplast DNA (ctDNA) has been constructed with the enzymes Sinai, PstI and PvulI. The ctDNA was found to be contained in a circular molecule of 148.5 kbp. In c o m m o n with many other higher plant ctDNAs, sugar beet ctDNA consists of two inverted repeat sequences of about 20.5 kbp separated by two single-copy regions of different sizes (about 23.2 and 84.3 kbp). Southern hybridization analyses indicated that the genes for rRNAs (23S + 16S) and the large subunit of ribulose 1,5-bisphosphate carboxylase were located in the inverted repeats and the large single-copy regions, respectively.
Introduction Higher plant chloroplasts contain their own DNA, which is present as a covalently closed circular molecule of 120-190 kbp. Chloroplast DNA (ctDNA) is thus well within the size range of DNA molecules directly amenable to restriction enzyme mapping techniques. Restriction maps o f ctDNAs from numerous plant species have now been published, and they provide a framework for the mapping of individual genes and for comparing the sequence organization of ctDNAs of different species (1, 10, 17). In sugar beet (Beta vulgaris L.), information available about ctDNA is limited. An electron microscopy (3) and restriction enzyme analysis (8, 11) have indicated its genome to be a circular molecule of 150-155 kbp in size, but nothing further is known about its detailed physico-chemical properties. This paper describes the construction of a restriction fragment map of sugar beet ctDNA. In addition, we have positioned the genes for the large subunit (LSU) of ribulose 1,5-bisphosphate carbox**To whom correspondenceshould be addressed.
ylase (RuBisCO) and the 23S and 16S ribosomal RNAs.
Materials and methods Preparation of ctDNA Leaves from 8 week old greenhouse-grown plants were used to prepare ctDNA from a male fertile strain (the maintainer for cytoplasmic male sterility), TK81-0. The ctDNA was isolated and purified as previously described (6).
Restriction enzyme analys& and Southern hybridization Restriction enzymes were purchased from Takara Shuzo Co. Ltd. and ctDNAs were digested with the enzymes according to the supplier's instructions. The procedure for physical mapping of restriction sites was as described by Herrmann et al. (4). It involved initial separation of restriction cleavage products of a first enzyme in a low-gellingtemperature agarose (BRL, USA) gel, subsequent
202 recovery and digestion of individual D N A bands with a second restriction enzyme. Two sets of molecular weight standards were included on each gel for size calibration; lambda D N A double digested with EcoRI and HindlII, or HaelII digests o f ~b × 174 RF DNA. Electrophoretic separation of the fragments, gel staining and Southern hybridization were performed essentially as described by Sugiura and Kusuda (15) and Shinozaki and Sugiura (14).
Electron microscopy The DNA-protein monolayers for electron microscopy were prepared by the aqueous and formamide method of Davis et al. (2). Grids were examined with a J E M 100S electron microscope at magnifications o f 4 0 0 0 - 2 0 0 0 0 times and the D N A molecules were traced as 10-fold-enlarged images with the aid of a digitizer. Calibration of the actual magnification was done with replica grating (2000 lines/mm). The ~b x 174 R F I I D N A (5 386 bp) was mounted on the same grids as an internal standard for size determinations of c t D N A molecules.
Results
Electron microscopy CsCl-ethidium bromide gradients of sugar beet c t D N A revealed the presence of two bands. Electron microscopic examination showed that the upper D N A band consisted mostly of linear molecules, but that a considerable amount of supercoiled and/or relaxed circular molecules were isolated from the lower band (Fig. 1). The contour length of the circular c t D N A (20 molecules) was measured as 45.3 + 1.5 #m, whereas that of × 174 R F I I DNA (20 molecules) was 1.58 ___ 0.08/zm. These values give 154 + 5.1 kbp or 101 ___ 3.4 × 106 daltons (d) for the sugar beet ctDNA. Our figure compares with the average contour length of 44.9 ___ 1.7 ttm reported by H e r r m a n n et al. (3) or the size of 99.9 + 2.0 × 106 d based upon SalGI restriction digests (11).
Fig. 1. Electron micrograph of a circular D N A molecule obtained from sugar ~b × 174 RFII DNA.
beet
chloroplasts.
Small
circles
are
Restriction fragment map Chloroplast D N A was digested with three restriction enzymes SmaI, PstI and PvulI, for physical mapping study. Each of these enzymes generated relatively few bands resulting in an easily resolvable banding pattern in single and double digests. Table 1 summarizes the sizes, numbers and multiplicity of all restriction fragments. The molecular weight of intact ctDNA was estimated by summing fragment sizes in each case and was found to be about 148.5 kbp. The strategy used in the ordering of restriction fragments has been detailed elsewhere (4, 13). Pri-
203 Table 1. Summary of sugar beet ctDNA fragments (kbp) produced by single and double digestions with the restriction endonucleases SmaI, PstI and PvulI. Band no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Sum
Sinai
Pstl
PvulI
21.7 18.6 14.7 14.0 9.8 8.4 7.1(3 x ) 6.7 5.4(2 x ) 3.7 2.64 2.33(2 × ) 2.10(2 x ) 1.86(2 x ) 1.71(2 x )
27.9 22.5(2 x ) 17.8 12.4 10.9 9.0 8.5 7.8 2.95 2.56 1.94 1.71
31.8 26.4 23.3 17.0 14.0 10.1 7.1 4.1(2x) 3.8 2.79(2 x ) 1.40
148.34
148.46
148.68
Sinai~ Pstl 18.6 9.8 9.3 9.0 8.4 7.8 7.6 6.8(2x) 5.4(3 x ) 5.3 3.7 3.4 2.95 2.91 2.56 2.33(2 x ) 2.25 2.17 2.10(2 x ) 1.94 1.86(2 x ) 1.71(3 x ) 1.55 0.54 0.47 0.33(2 x ) 148.41
SmaI/ PvulI 18.6 14.7 14.0 9.8 9.6 8.4 7.1(3 × ) 4.3(2×) 3.7(2 x ) 3.4(2 x ) 3.1 2.95 2.64 2.33(2 x ) 1.86(2 × ) 1.55(2 x ) 1.40 1.24(4x) 0.62 0.54(2 x ) 0.47(2 x ) 0.33
148.70
PstI/ PvulI 21.7 14.0 10.9 9.3 9.0 8.5 7.8 7.1 6.8(2 × ) 5.6 5.3 4.0(2 x ) 3.8(2 × ) 3.6 2.79(2 × ) 2.56 1.94 1.71(2x) 1.40 1.24 0.47
148.61
Numbers in brackets refer to multiple copies.
m a r y f r a g m e n t s w e r e e x t r a c t e d f r o m gel slices, a n d further digested with a second enzyme. Single and double digests of total ctDNA were included in parallel t r a c k s f o r t h e b a n d i d e n t i f i c a t i o n . F o r i n !stance, S m a - 1 (S1) a n d P s t - 5 ( P 5 ) f r a g m e n t s overlapped v/a a common subfragment Sma/Pst-4 ( S P 4 ) . T h e o t h e r e n d - s u b f r a g m e n t o f P 5 w a s SP17, w h i c h w a s a l s o a n e n d - s u b f r a g m e n t o f STa (Tab l e 1). S 7 a c o n t a i n e d t w o a d d i t i o n a l s u b f r a g m e n t s SP14 and SP20:SP14 was found in P2a, while SP20 was equivalent to Pll. Additionally, P2a carried a s u b f r a g m e n t SP1 i n c o m m o n w i t h $2. T h u s , t h e orders of Sinai and PstI fragments in this region w e r e d e t e r m i n e d t o b e $ 2 - STa - S1 a n d P 2 a P l l - P 5 (Fig. 2). We have constructed a physical map for the Sinai, PstI and PvuII fragments, based upon the reciprocal experiments involving sequential digestion of individual fragments with the endonuclease
Fig. 2. Restriction fragment map of sugar beet ctDNA, showing the two inverted repeats (IR) and the locations of the genes for 23S and 16S rRNAs and the large subunit (LSU) of RuBisCO. Fragments are labelled in order of decreasing size (see Table 1).
204 pairs. Our mapping data demonstrate that sugar beet ctDNA is circular and contains two identical sequences in an inverted orientation (Fig. 2). The inverted repeat sequences are at least 20.5 kbp in size (S7b + $9 + S12 + S13 + S14 + S15). A confirming result of the restriction map was obtained from the hybridization carried out with a tobacco probe. In tobacco ctDNA, the Bam-2 fragment is known to occupy most of the small singlecopy region. This fragment hybridized to sugar beet fragments of $3 and $6 which were considered to be located in a small single-copy region (data not shown). Localization o f rRNA and L S U genes
Chloroplast genes have been mapped on the sugar beet chloroplast chromosome using probes from tobacco. A mixture of tobacco 23S and 16S rRNAs hybridized exclusively to $9, S12, S15, P1 and P2b fragments (data not shown). As shown in Fig. 2, all these fragments are parts of the inverted repeat. The LSU gene probe hybridized with S7a, P2a and SP14 fragments (data not shown). The approximate positions of these genes are indicated in the restriction map (Fig. 2).
Discussion From the present data, we have concluded that sugar beet chloroplasts contain circular DNA molecules of approximately 148.5 kbp, organized into four distinct components. The two inverted repeats (each about 20.5 kbp) are separated by a small (23.2 kbp) and a large (84.3 kbp) single-copy regions. Southern hybridization analyses indicated the rRNA genes and the LSU gene to be situated at the inverted repeats and the large single-copy regions, respectively. Thus, sugar beet chloroplast genome proves to be similar in size, conformation, and physical and gene organization, to ctDNAs from a number of angiosperms (1, 10, 17). In Nicotiana chloroplast genomes, the most frequently observed variations are point mutations and deletions or insertions which are located within several regions scattered along the chloroplast chromosome (5, 12, 16). Beta species also exhibit a high degree of variability of ctDNAs (7, 9). Comparative studies on the restriction maps of ctDNAs among
many Beta species will help to elucidate the nature of this variation and the biological implication of structural changes in view of molecular evolution. This approach is currently in progress.
Acknowledgements We thank Professor E. Shikata of Hokkaido University for his kind help. This work was supported in part by Grants in-Aid from the Ministry of Education, Science and Culture, Japan.
References 1. Bedbrook JR, Kolodner R: The structure of chloroplast DNA. Ann Rev Plant Physiol 30:593-620, 1979. 2. Davis RW, Simon M, Davidson N: Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acid. In: Grossmann L, Moldave K (eds) Methods of enzymology. Vol 21. Academic Press, New York, 1971, lap 413-428. 3. Herrmann RG, Bohnert H-J, Kowallik KV, Schmitt JM: Size, conformation and purity of chloroplast DNA of some higher plants. Biochim Biophys Acta 378:305- 317, 1975. 4. Herrmann RG, Whitfeld PR, Bottomley W: Construction of a SalI/Pstl restriction map of spinach chloroplast DNA using low-gelling-temperature-agarose electrophoresis. Gene 8:179- 191, 1980. 5. Kung SD, Zhu YS, Shen GF: Nicotiana chloroplast genome. 3. Chloroplast DNA evolution. Theor Appl Genet 61:73-79, 1982. 6. Mikami T, Sugiura M, Kinoshita T: Molecular heterogeneity in mitochondrial and chloroplast DNAs from normal and male sterile cytoplasms in sugar beets. Curr Genet 8:319- 322, 1984. 7. Mikami T, Kishima Y, Sugiura M, Kinoshita T: Chloroplast DNA diversity in the cytoplasms of sugar beet and its related species. Plant Sci Lett 36:231- 235, 1984. 8. Mikami T, Shinozaki K, Sugiura M, Kinoshita T: Characterization of chloroplast DNA from sugar beet with normal and male sterile cytoplasms. Jpn J Genet 59:497-504, 1984. 9. Mikami T, Kishima Y, Sugiura M, Kinoshita T: Organelle genome diversity in sugar beet with normal and different sources of male sterile cytoplasms. Theor Appl Genet 71:166- 171, 1985. 10. Palmer JD: Comparative organization of chloroplast genomes. Ann Rev Genet 19:325-354, 1985. 11. Powling A, Ellis THN: Studies on the organelle genomes of sugar beet with male-fertile and male-sterile cytoplasms. Theor Appl Genet 65:323- 328, 1983. 12. Salts Y, Herrmann RG, Peleg N, Lavi U, Izhar S, Frankel R, Beckmann JS: Physical mapping of plastid DNA variation among eleven Nicotiana species. Theor Appl Genet 69:1 - 14, 1984.
205 13. Seyer P, Kowallik KV, Herrmann RG: A physical map of Nicotiana tabacum plasmid DNA including the location of structural genes for ribosomal RNAs and the large subunit of ribulose ,bisphosphate carboxylase/oxygenase. Curr Genet 3:189-204, 1981. 14. Shinozaki K, Sugiura M: The nucleotide sequence of the tobacco chloroplast gene for the large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase. Gene 20:91 - 102, 1982. 15. Sugiura M, Kusuda J: Molecular cloning of tobacco chlo-
roplast ribosomal RNA genes. Mol Gen Genet 172:137- 141, 1979. 16. Tassopulu D, Kung SD: Nicotiana chloroplast genome. 6. Deletion and hot spot - a proposed origin of the inverted repeats. Theor Appl Genet 67:185-193, 1984. 17." Whitfeld PR, Bottomley M" Organization and structure of chloroplast genes. Ann Rev Plant Physiol 34:279-310, 1983. Received 26 February; accepted 10 June 1986.
201
Restriction fragment map of sugar beet ( B e t a vulgaris L . ) chloroplast D N A Yuji Kishima, 1 Tetsuo Mikami, 1 Takeo Harada, 1,* Kazuo Shinozaki, 2 Masahiro Sugiura 2 & Toshiro Kinoshital, ** 1Plant Breeding Institute, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan; 2Center for Gene Research, Nagoya University, Chikusa, Nagoya 464, Japan; *Present address: Hokkaido Central Agricultural Experiment Station, Naganuma 069-13, Japan
Keywords: Beta vulgaris, chloroplast DNA, electron microscopy, restriction fragment map, sugar beet
Summary A restriction endonuclease fragment map of sugar beet chloroplast DNA (ctDNA) has been constructed with the enzymes Sinai, PstI and PvulI. The ctDNA was found to be contained in a circular molecule of 148.5 kbp. In c o m m o n with many other higher plant ctDNAs, sugar beet ctDNA consists of two inverted repeat sequences of about 20.5 kbp separated by two single-copy regions of different sizes (about 23.2 and 84.3 kbp). Southern hybridization analyses indicated that the genes for rRNAs (23S + 16S) and the large subunit of ribulose 1,5-bisphosphate carboxylase were located in the inverted repeats and the large single-copy regions, respectively.
Introduction Higher plant chloroplasts contain their own DNA, which is present as a covalently closed circular molecule of 120-190 kbp. Chloroplast DNA (ctDNA) is thus well within the size range of DNA molecules directly amenable to restriction enzyme mapping techniques. Restriction maps o f ctDNAs from numerous plant species have now been published, and they provide a framework for the mapping of individual genes and for comparing the sequence organization of ctDNAs of different species (1, 10, 17). In sugar beet (Beta vulgaris L.), information available about ctDNA is limited. An electron microscopy (3) and restriction enzyme analysis (8, 11) have indicated its genome to be a circular molecule of 150-155 kbp in size, but nothing further is known about its detailed physico-chemical properties. This paper describes the construction of a restriction fragment map of sugar beet ctDNA. In addition, we have positioned the genes for the large subunit (LSU) of ribulose 1,5-bisphosphate carbox**To whom correspondenceshould be addressed.
ylase (RuBisCO) and the 23S and 16S ribosomal RNAs.
Materials and methods Preparation of ctDNA Leaves from 8 week old greenhouse-grown plants were used to prepare ctDNA from a male fertile strain (the maintainer for cytoplasmic male sterility), TK81-0. The ctDNA was isolated and purified as previously described (6).
Restriction enzyme analys& and Southern hybridization Restriction enzymes were purchased from Takara Shuzo Co. Ltd. and ctDNAs were digested with the enzymes according to the supplier's instructions. The procedure for physical mapping of restriction sites was as described by Herrmann et al. (4). It involved initial separation of restriction cleavage products of a first enzyme in a low-gellingtemperature agarose (BRL, USA) gel, subsequent
202 recovery and digestion of individual D N A bands with a second restriction enzyme. Two sets of molecular weight standards were included on each gel for size calibration; lambda D N A double digested with EcoRI and HindlII, or HaelII digests o f ~b × 174 RF DNA. Electrophoretic separation of the fragments, gel staining and Southern hybridization were performed essentially as described by Sugiura and Kusuda (15) and Shinozaki and Sugiura (14).
Electron microscopy The DNA-protein monolayers for electron microscopy were prepared by the aqueous and formamide method of Davis et al. (2). Grids were examined with a J E M 100S electron microscope at magnifications o f 4 0 0 0 - 2 0 0 0 0 times and the D N A molecules were traced as 10-fold-enlarged images with the aid of a digitizer. Calibration of the actual magnification was done with replica grating (2000 lines/mm). The ~b x 174 R F I I D N A (5 386 bp) was mounted on the same grids as an internal standard for size determinations of c t D N A molecules.
Results
Electron microscopy CsCl-ethidium bromide gradients of sugar beet c t D N A revealed the presence of two bands. Electron microscopic examination showed that the upper D N A band consisted mostly of linear molecules, but that a considerable amount of supercoiled and/or relaxed circular molecules were isolated from the lower band (Fig. 1). The contour length of the circular c t D N A (20 molecules) was measured as 45.3 + 1.5 #m, whereas that of × 174 R F I I DNA (20 molecules) was 1.58 ___ 0.08/zm. These values give 154 + 5.1 kbp or 101 ___ 3.4 × 106 daltons (d) for the sugar beet ctDNA. Our figure compares with the average contour length of 44.9 ___ 1.7 ttm reported by H e r r m a n n et al. (3) or the size of 99.9 + 2.0 × 106 d based upon SalGI restriction digests (11).
Fig. 1. Electron micrograph of a circular D N A molecule obtained from sugar ~b × 174 RFII DNA.
beet
chloroplasts.
Small
circles
are
Restriction fragment map Chloroplast D N A was digested with three restriction enzymes SmaI, PstI and PvulI, for physical mapping study. Each of these enzymes generated relatively few bands resulting in an easily resolvable banding pattern in single and double digests. Table 1 summarizes the sizes, numbers and multiplicity of all restriction fragments. The molecular weight of intact ctDNA was estimated by summing fragment sizes in each case and was found to be about 148.5 kbp. The strategy used in the ordering of restriction fragments has been detailed elsewhere (4, 13). Pri-
203 Table 1. Summary of sugar beet ctDNA fragments (kbp) produced by single and double digestions with the restriction endonucleases SmaI, PstI and PvulI. Band no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Sum
Sinai
Pstl
PvulI
21.7 18.6 14.7 14.0 9.8 8.4 7.1(3 x ) 6.7 5.4(2 x ) 3.7 2.64 2.33(2 × ) 2.10(2 x ) 1.86(2 x ) 1.71(2 x )
27.9 22.5(2 x ) 17.8 12.4 10.9 9.0 8.5 7.8 2.95 2.56 1.94 1.71
31.8 26.4 23.3 17.0 14.0 10.1 7.1 4.1(2x) 3.8 2.79(2 x ) 1.40
148.34
148.46
148.68
Sinai~ Pstl 18.6 9.8 9.3 9.0 8.4 7.8 7.6 6.8(2x) 5.4(3 x ) 5.3 3.7 3.4 2.95 2.91 2.56 2.33(2 x ) 2.25 2.17 2.10(2 x ) 1.94 1.86(2 x ) 1.71(3 x ) 1.55 0.54 0.47 0.33(2 x ) 148.41
SmaI/ PvulI 18.6 14.7 14.0 9.8 9.6 8.4 7.1(3 × ) 4.3(2×) 3.7(2 x ) 3.4(2 x ) 3.1 2.95 2.64 2.33(2 x ) 1.86(2 × ) 1.55(2 x ) 1.40 1.24(4x) 0.62 0.54(2 x ) 0.47(2 x ) 0.33
148.70
PstI/ PvulI 21.7 14.0 10.9 9.3 9.0 8.5 7.8 7.1 6.8(2 × ) 5.6 5.3 4.0(2 x ) 3.8(2 × ) 3.6 2.79(2 × ) 2.56 1.94 1.71(2x) 1.40 1.24 0.47
148.61
Numbers in brackets refer to multiple copies.
m a r y f r a g m e n t s w e r e e x t r a c t e d f r o m gel slices, a n d further digested with a second enzyme. Single and double digests of total ctDNA were included in parallel t r a c k s f o r t h e b a n d i d e n t i f i c a t i o n . F o r i n !stance, S m a - 1 (S1) a n d P s t - 5 ( P 5 ) f r a g m e n t s overlapped v/a a common subfragment Sma/Pst-4 ( S P 4 ) . T h e o t h e r e n d - s u b f r a g m e n t o f P 5 w a s SP17, w h i c h w a s a l s o a n e n d - s u b f r a g m e n t o f STa (Tab l e 1). S 7 a c o n t a i n e d t w o a d d i t i o n a l s u b f r a g m e n t s SP14 and SP20:SP14 was found in P2a, while SP20 was equivalent to Pll. Additionally, P2a carried a s u b f r a g m e n t SP1 i n c o m m o n w i t h $2. T h u s , t h e orders of Sinai and PstI fragments in this region w e r e d e t e r m i n e d t o b e $ 2 - STa - S1 a n d P 2 a P l l - P 5 (Fig. 2). We have constructed a physical map for the Sinai, PstI and PvuII fragments, based upon the reciprocal experiments involving sequential digestion of individual fragments with the endonuclease
Fig. 2. Restriction fragment map of sugar beet ctDNA, showing the two inverted repeats (IR) and the locations of the genes for 23S and 16S rRNAs and the large subunit (LSU) of RuBisCO. Fragments are labelled in order of decreasing size (see Table 1).
204 pairs. Our mapping data demonstrate that sugar beet ctDNA is circular and contains two identical sequences in an inverted orientation (Fig. 2). The inverted repeat sequences are at least 20.5 kbp in size (S7b + $9 + S12 + S13 + S14 + S15). A confirming result of the restriction map was obtained from the hybridization carried out with a tobacco probe. In tobacco ctDNA, the Bam-2 fragment is known to occupy most of the small singlecopy region. This fragment hybridized to sugar beet fragments of $3 and $6 which were considered to be located in a small single-copy region (data not shown). Localization o f rRNA and L S U genes
Chloroplast genes have been mapped on the sugar beet chloroplast chromosome using probes from tobacco. A mixture of tobacco 23S and 16S rRNAs hybridized exclusively to $9, S12, S15, P1 and P2b fragments (data not shown). As shown in Fig. 2, all these fragments are parts of the inverted repeat. The LSU gene probe hybridized with S7a, P2a and SP14 fragments (data not shown). The approximate positions of these genes are indicated in the restriction map (Fig. 2).
Discussion From the present data, we have concluded that sugar beet chloroplasts contain circular DNA molecules of approximately 148.5 kbp, organized into four distinct components. The two inverted repeats (each about 20.5 kbp) are separated by a small (23.2 kbp) and a large (84.3 kbp) single-copy regions. Southern hybridization analyses indicated the rRNA genes and the LSU gene to be situated at the inverted repeats and the large single-copy regions, respectively. Thus, sugar beet chloroplast genome proves to be similar in size, conformation, and physical and gene organization, to ctDNAs from a number of angiosperms (1, 10, 17). In Nicotiana chloroplast genomes, the most frequently observed variations are point mutations and deletions or insertions which are located within several regions scattered along the chloroplast chromosome (5, 12, 16). Beta species also exhibit a high degree of variability of ctDNAs (7, 9). Comparative studies on the restriction maps of ctDNAs among
many Beta species will help to elucidate the nature of this variation and the biological implication of structural changes in view of molecular evolution. This approach is currently in progress.
Acknowledgements We thank Professor E. Shikata of Hokkaido University for his kind help. This work was supported in part by Grants in-Aid from the Ministry of Education, Science and Culture, Japan.
References 1. Bedbrook JR, Kolodner R: The structure of chloroplast DNA. Ann Rev Plant Physiol 30:593-620, 1979. 2. Davis RW, Simon M, Davidson N: Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acid. In: Grossmann L, Moldave K (eds) Methods of enzymology. Vol 21. Academic Press, New York, 1971, lap 413-428. 3. Herrmann RG, Bohnert H-J, Kowallik KV, Schmitt JM: Size, conformation and purity of chloroplast DNA of some higher plants. Biochim Biophys Acta 378:305- 317, 1975. 4. Herrmann RG, Whitfeld PR, Bottomley W: Construction of a SalI/Pstl restriction map of spinach chloroplast DNA using low-gelling-temperature-agarose electrophoresis. Gene 8:179- 191, 1980. 5. Kung SD, Zhu YS, Shen GF: Nicotiana chloroplast genome. 3. Chloroplast DNA evolution. Theor Appl Genet 61:73-79, 1982. 6. Mikami T, Sugiura M, Kinoshita T: Molecular heterogeneity in mitochondrial and chloroplast DNAs from normal and male sterile cytoplasms in sugar beets. Curr Genet 8:319- 322, 1984. 7. Mikami T, Kishima Y, Sugiura M, Kinoshita T: Chloroplast DNA diversity in the cytoplasms of sugar beet and its related species. Plant Sci Lett 36:231- 235, 1984. 8. Mikami T, Shinozaki K, Sugiura M, Kinoshita T: Characterization of chloroplast DNA from sugar beet with normal and male sterile cytoplasms. Jpn J Genet 59:497-504, 1984. 9. Mikami T, Kishima Y, Sugiura M, Kinoshita T: Organelle genome diversity in sugar beet with normal and different sources of male sterile cytoplasms. Theor Appl Genet 71:166- 171, 1985. 10. Palmer JD: Comparative organization of chloroplast genomes. Ann Rev Genet 19:325-354, 1985. 11. Powling A, Ellis THN: Studies on the organelle genomes of sugar beet with male-fertile and male-sterile cytoplasms. Theor Appl Genet 65:323- 328, 1983. 12. Salts Y, Herrmann RG, Peleg N, Lavi U, Izhar S, Frankel R, Beckmann JS: Physical mapping of plastid DNA variation among eleven Nicotiana species. Theor Appl Genet 69:1 - 14, 1984.
205 13. Seyer P, Kowallik KV, Herrmann RG: A physical map of Nicotiana tabacum plasmid DNA including the location of structural genes for ribosomal RNAs and the large subunit of ribulose ,bisphosphate carboxylase/oxygenase. Curr Genet 3:189-204, 1981. 14. Shinozaki K, Sugiura M: The nucleotide sequence of the tobacco chloroplast gene for the large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase. Gene 20:91 - 102, 1982. 15. Sugiura M, Kusuda J: Molecular cloning of tobacco chlo-
roplast ribosomal RNA genes. Mol Gen Genet 172:137- 141, 1979. 16. Tassopulu D, Kung SD: Nicotiana chloroplast genome. 6. Deletion and hot spot - a proposed origin of the inverted repeats. Theor Appl Genet 67:185-193, 1984. 17." Whitfeld PR, Bottomley M" Organization and structure of chloroplast genes. Ann Rev Plant Physiol 34:279-310, 1983. Received 26 February; accepted 10 June 1986.
