Oenotis
7
CUrrt © Springer-Verlag 1983
Mapping the Chloroplast DNA of Viciafaba Kenton Ko, Neil A. Straus, and John P. Williams Department of Botany, University of Toronto, Toronto, Ontario, Canada, M5S 1A1
Summary. A complete clone bank representing the chloroplast DNA from Vicia faba has been constructed. A total of 15 fragments (10 Pstl, 1 Pstl-EcoR1 and 4 Sall fragments) were inserted into the vector pBR322 and transformed into the E coli strain HB101. The cloned fragments were used as the main tools in constructing the physical map of Vicia faba for the restriction endonucleases Pstl, Kpnl and Xhol. The identity of the cloned fragments was demonstrated by restriction analysis and blot hybridization. The information generated was used to construct the map. The 16S and 23S rRNA genes and the gene for the large subunit ofribulose1,5-bisphosphate carboxylase have been positioned on the map using heterologous probes. The orientation of the gene for the large subunit of RuBP carboxylase has also been determined. Key words: Chloroplast DNA clone bank - Physical mapping - Gene mapping
Introduction The chloroplast genome of most vascular plants consists of a single circular molecule containing 130-150 kilobase pairs (kb) (Bedbrook and Kol0dner 1979). A 2 0 - 2 5 kbp sequence of DNA that includes the ribosomal RNA genes has been shown in a number of species to be repeated once and arranged in an inverted orientation. The presence of the inverted repeated region has been verified by restriction endonuclease mapping for Zea mays, corn (Bedbrook and Bogorad 1976), Spinacea oleracea, spinach (Herrmann et al. 1980), Spirodela
Offprint requests to: K. Ko
oligorrhiza (van Ee et al. 1980), Sinapsis alba, mustard, (Link et al. 1981), Petunia hybrida, petunia, (Bovenberg et al. 1981), Oenothera parviflora (Gordon et al. 1981), Nieotiana tabaeum, tobacco (Fluhr and Edelman 1981), Vigna radiata, mung bean (Palmer and Thompson 1981 b), Pennisetum americanum (Rawson et al. 1981), Atriplex triangularis (Palmer 1982) and Cucumis sativa, cucumber (Palmer 1982). The chloroplast chromosome of at least three species of the family Fabaceae (Leguminosae) differ from the regular pattern of sequence arrangement. Vicia faba, broad bean (Koller and Delius 1980), Pisum sativum, pea (Palmer and Thompson 1981; Chu et al. 1981), and L3cer arietum (Chu and Tewari 1982) have smaller genomes which lack the inverted region and contain only one copy of the genes for chloroplast ribosomal RNA (rRNA). In this study we have physically mapped the chloroplast chromosome of Vicia faba with three restriction endonucleases and created a complete gene library in 15 cloned fragments. Our map confirms the basic arrangement of a Kpnl map published earlier (KoUer and Delius 1980) but includes two fragments that were overlooked in the previous study. In addition we have positioned the genes for the large subunit of ribulose1,5-bisphosphate carboxylase and the 16S and 23S ribosomal RNA.
Materials and Methods
Chloroplast DNA Isolation. Chloroplast DNA was isolated from 4 week old Vieiafaba (L. cv. Broad Windsor) plants by a modification of the method of Manning et al. (1971). The plants were destarched by keeping them in the dark for 12-36 h prior to isolation. The leaves (150-200 g) were quickly frozen in liquid N2 and blended in a Waring blender by the method of Rhodes and Kung (1981). The liquid N2 was allowed to evaporate. When
256 the blended leaves reached - 5 °C, approximately 900 ml of ice cold isolation buffer A [0.33 M sorbitol, 10 mM Tris pH 8.0, 50 mM ethylenediaminetetraacetic acid (EDTA), 5 mM 2mercaptoethanol, 0.1% bovine serum albumin (BSA) (w/v)l was added. The extract was filtered through two layers of cheesecloth and one layer of nylon mesh (20 /~m pore size). The filtrate was centrifuged at 1,500 x g for 15 min at 4 °C. The pellet was resuspended in 50 ml of ice cold isolation buffer B [10% sucrose (w/v), 10 mM Tris pH 8.0, 50 mM EDTA, 5 mM 2-mercaptoethanol, 0.1% BSA (w/v)]. Then 120 ml of ice cold isolation buffer C [75% sucrose (w/v), 10 mM Tris pH 8.0, 50 mM EDTA, 5 mM 2-mercaptoethanol, 0.1% BSA (w/v)l was added to give a final sucrose concentration of 5 5 56%. The mixture was centrifuged at 16,000 x g for 50 rain at 4 ° C. The floating chloroplast layer was collected and resuspended in isolation buffer B. The suspension was centrifuged at 1,000 x g for 20 min at 4 °C and the resulting pellet was resuspended in 6 ml of 10 mM Tris pH 8.0, 10 mM EDTA. The chloroplasts were lysed by adding 750 td of 20% sodium lauryl sulphate (SLS). Then 400 #1 of 5 M NaC1 was added and the lysate was deproteinized by extracting with equal volumes of Tris buffered phenol, followed by CHC13:isoamyl alcohol [24: l(v/v)l. The DNA was precipitated by the addition of two volumes of cold ethanol and collected by spooling onto a glass rod. The DNA was redissolved in 10 mM Tris pH 8.0, 1 mM EDTA. RNAse A was added to a final concentration of 100/~g/ml and the mixture was incubated at 37 °C for 1 h. The solution was deproteinizedwith sequentialphenol and CHC13 extractions as described earlier. The DNA was reprecipitated and redissolved in distilled H20.
Plasmid Isolation. Plasmids were isolated by a modification of the method of Mural et al. (1979). Plasmid-containing bacteria were grown in M9 minimal medium to an optical density of 0.6 at 650 nm. Two ml of 100 mg/ml chloramphenicol was added to 1 1 of culture and the culture was incubated overnight at 37 °C. The cells were harvested by eentrifugation at 700 x g for 10 min and resuspended in 40 ml of 10% sucrose (w/v), 50 mM Tris pH 8.0 Sixteen ml of 0.2 M EDTA pH 8.0 and 8 ml of lysozyme (5 mg/ml) was added and the mixture kept on ice for 20 rain. The cells were gently lysed by adding 4 ml 2% Sarkosyl. The lysate was clarified by centrifugation for 1.5 h at 13,000 rpm in a HB-4 rotor at 4 °C. The supematant was deproteinized as described in the chloroplast DNA isolation. The DNA was ethanol precipitated and recovered by centrifugation at 12,000 x g for 20 rain at 4 °C. Plasmid DNA was further purified by centrifugation to equilibrium in cesium chlorideethidium bromide gradients as described by Davis et al. (1980).
Restriction Endonuclease
Analysis. Chloroplast DNA was digested with various restriction endonucleases according to the suppliers instructions (Bethesda Research Laboratories, Inc.; New England Biolabs Inc.). Digested DNA's were electrophoresed in 0.7% horizontally submerged agarose gel for 1 8 - 2 0 h at 1.5 volts per cm of gel. The electrophoresis buffer was 80 mM Tris, pH 8.0, 10 mM sodium acetate, 2 mM EDTA. The gels were stained with ethidium bromide and visualized on a UV transilluminator.
K. Ko et al.: Mapping the Chloroplast DNA of Vicia faba transformants and their plasmids isolated from small cultures were analyzed by restriction endonucleases and agarose gel electrophoresis. The authenticity of the recombinant plasmids containing chloroplast DNA fragments was confirmed by blot hybridization to total restricted chloroplast DNA.
Radioactive Labelling of Probes. The recombinant plasmids and pJZA4 were labelled with a32P dCTP (Amersham Corp.) according to the supplier's instructions in a nick translation kit purchased from Bethesda Research Laboratories Inc. The plasmid, pJZA4 was a generous gift from Dr. S. D. Kung. E. coli rRNA was end labelled with 732p ATP (Amersham Corp.) according to Maxam and Gilbert (1980). The E. coli rRNAs were a generous gift from G. P. Singh.
Blot Hybridization. DNA fragments were transferred onto nitrocellulose sheets (Schleicher and Schuell - BA85) according to Southern (1975). The nitrocellulose filters were hybridized with 32p probes according to Fluhr and Edelman (1981).
Results and Discussion Restriction Endonuclease Analysis C h l o r o p l a s t D N A i s o l a t e d f r o m Vicia faba was digested w i t h various r e s t r i c t i o n e n z y m e s . T h e e l e c t r o p h o r e t i c p a t t e r n s o f t h e digestion p r o d u c t s are p r e s e n t e d in Fig. 1. S o m e o f t h e e n z y m e s s u c h as B a m H I and HindlII, digested t h e c h l o r o p l a s t D N A i n t o a large n u m b e r o f f r a g m e n t s (Fig. 1 a n d 2) while t h e r e m a i n i n g e n z y m e s p r o d u c e d a m o d e r a t e n u m b e r o f f r a g m e n t s (Fig. 2). T h e r e s t r i c t i o n f r a g m e n t sizes p r o d u c e d b y e n z y m e s K p n l , PsH, Sall a n d X h o l are c o m p i l e d in T a b l e 1. T h e f r a g m e n t sizes were e s t i m a t e d using HindlII digested l a m b d a D N A f r a g m e n t s as m o l e c u l a r size m a r k e r s (Maniatis e t al., 1975). K p n l , Pstl a n d X h o l were s e l e c t e d as t h e p r i m a r y m a p p i n g e n z y m e s because t h e y p r o d u c e d a m o d e r a t e n u m b e r o f discrete f r a g m e n t s t h a t were resolvable b y gel e l e c t r o p h o r e s i s . C h l o r o p l a s t D N A was also a n a l y z e d w i t h t w o pairs o f r e s t r i c t i o n e n z y m e s : P s t l - K p n l and P s t l - X h o l . These d o u b l e digestion p a t t e r n s are s h o w n in Fig. 3 a n d t h e f r a g m e n t sizes are c o m p i l e d in T a b l e 1. B o t h d o u b l e digestions p r o d u c e d a set o f 2 4 discrete fragments. T h e t o t a l m o l e c u l a r size o f t h e c h l o r o p l a s t D N A was e s t i m a t e d b y a d d i n g t h e f r a g m e n t sizes in b o t h single a n d d o u b l e digestions. These results are c o m p u t e d as t o t a l s in T a b l e 1. T h e e s t i m a t e d average overall size was
DNA Cloning. Pstl, Sail and Pst-EcoR1 restriction fragments of chloroplast DNA were inserted into the appropriate sites of pBR322 with T 4 DNA ligase by the method of Dugaiczyk et al. (1975). The recombinant plasmids were transformed into the E. coli strain HB101 according to Cohen et al. (1972). Colonies containing plasmids with inserts of chloroplast DNA were detected by their appropriate antibiotic phenotypes. Selected
Fig. 1. Gel electrophoresis of restriction endonuclease digests • of Vicia faba chloroplast DNA. Restriction patterns are shown for Smal (lane 2), BamHI (lane 3), HindlI1 (lane 4), Pstl (lane 5), Sall (lane 6) and HindlfI digested lambda DNA (lanes 1 and 7). The fragment sizes for the Sall digest are compiled in Table 1. Fragment sizes are given in kilobase pairs
K. Ko et al.: Mapping the Chloroplast DNA of Vicia faba
257
Table 1. Restriction enzyme analysis of chloroplast DNA
Pst l 35.0 20.0 15.5 14.5 13.0 10.0 6.3 5.2 1.8 1.1 0.6
(P1) (P2) (P3) (P4) (P5) (P6) (P7) (P8) (P9) (P10) (Pll)
123,000
Xho l
Kpnl
Sail
20.5 (X1) 19.5 (X2) 16.0 (X3) 15.0 (X4) 14.0 (X5) 12.0 (X6) 10.5 (X7) 4.2 (X8) 3.2 (X9) 3.0 (X10a & b) 1.25 ( X l l ) 0.6 (X12)
24.5 23.5 20.0 12.5 10.5 8.1 5.0 4.3 4.1 3.0 1.1 0.6
122,650
122,200
(K1) (K2) (K3) (K4) (KS) (K6) (K7a & b) (K8) (K9) (K10) (Kll) (K12)
31.0 16.5 12.5 10.0 8.4 1.6
(Sla & b) ($2) (S3a & b) ($4) ($5) (S6)
123,800
Pstl-Kpnl
Pstl-Xho l
20.5 12.5 11.5 10.0 8.0 (2x) 7.0 6.2 5.2 4.8 4.5 4.3 3.6 3.1 (2x) 2.6 (2x) 1.8 1.0 (2x) 0.9 0.76 0.7 0.6
14.5 12.0 10.0 9.4 (3x) 8.1 7.2 (2x) 5.2 (2x) 3.5 3.2 3.0 (3x) 2.1 1.8 1.3 1.1 0.9 0.6 (3x)
124,260
122,300
Native chloroplast DNA was digested with the restriction endonucleases indicated. Fragment sizes were determined using HindIII digested lambda DNA as size markers. The fragments produced are given in kilobase pairs. The totals are recorded in base pairs. The brackets in Pstl, Xhol, Kpnl and Sall indicate fragment designations. The brackets in Pstl-Kpnl and Pstl-Xhol indicate the number of co-migrating bands
Fig. 2. Gel electrophoretic patterns of Vicia faba chloroplast DNA digested with the three mapping endonucleases. Restriction patterns are shown for Pstl (lane 2), Kpnl (lane 3) and Xhol (lane 4). HindIII digest of lambda DNA is the size markers in lane 1. Fragment sizes are given in kilobase pairs
258
K. Ko et al.: Mapping the Chloroplast DNA of Viciafaba Construction o f Chloroplast DNA Clone Bank
Fig. 3. Double digestion patterns of Vicia faba chloroplast DNA. The double digestion patterns are shown for Pstl-Xhol (lane 2), Pstl (lane 3), Pstl-Kpnl (lane 4) and Xhol (lape 5). HindlII digest of lambda DNA is in lane 1. Fragment sizes are given in kilobase pairs
123,000 base pairs. The size estimated in this study is comparable to the size reported by Koller and Delius (1980). They reported 79.8 x 106 daltons or approximately 120,000 base pairs. However, they have overlooked two K p n l fragments totalling 1,700 base pairs or approximately 1.1 x 106 daltons. With these two K p n l fragments, the total size should be 80.9 x 106 daltons or about 122,000 base pairs. Blot hybridization confirmed the presence of these fragments.
A clone bank representing the entire chloroplast genome from V. faba was constructed. This clone bank was used as a source of discrete restriction fragments for physical mapping. In addition, it is a source of chloroplast genes to study the molecular genetics of photosynthesis. Pstl was selected as the primary cloning enzyme due to the fragments produced. The fragments generated were all well resolved; none of the eleven fragments were comigrating. Ten out of eleven of the Pstl fragments were cloned into the single Pstl site within the ampicillin resistant gene of pBR322 (Bolivar et al. 1977). Figure 4 shows the electrophoretic profile of all ten of these clones after digestion with Pstl. Approximately 73% of the chloroplast DNA was cloned into the Pstl site of pBR322. However, the largest Pstl fragment (35 kb) was not recovered, probably because the fragment is simply too large for direct cloning. In order to bring the fragment to a workable size, the Pstl fragment was isolated on a 10-30% sucrose gradient and partially digested with EcoR1 in the hope of cutting the majority of the fragments in half. The partial fragments were inserted into pBR322 that had been digested with both Pstl and EcoR1. One third of the fragment was cloned by this strategy, increasing the portion of the chloroplast genome cloned to 83%. The cloned fragment was identified by double digestion and blot hybridization (see mapping results). In order to clone the remaining part of the chloroplast genome, a partial clone bank in pBR322 of Sall fragments of V. faba chloroplast DNA was created and those fragments that overlap the Pstl fragments were selected (see mapping section). The Sall fragments that overlap Pstl fragments correspond to the Sall fragments 3a, 2, 5 and 6 of Koller and Delius (1980). The Vicia faba chloroplast DNA clone bank is complete in fifteen cloned fragments (see Table 2). Two areas of overlap exist; one between the cloned fragment
Fig. 4. Gel electrophoretic analysis of the ten Pstl cloned fragments. Lanes 1 and 16 contain HindIII digested lambda DNA. Lanes 2 and 15 contain Pstl digested chloroplast DNA: Lanes 3 and 14 contain Pstl digested pBR322. Lanes 4 through 13 contain Pstl digested clones from pVFP2 through pVFP11 in sequential order
K. Ko et al.: Mapping the Chloroplast DNA of Viciafaba
259
Table 2. ViciaFaba chloroplast DNA clone bank pBR322 Clone designation
Fragment cloned
pVFP2 pVFP3 pVFP4 pVFP5 pVFP6 pVFP7 pVFP8 pVFP9 pVFP10 pVFP11 pVFPla pVFS2 pVFS3a pVFS5/6 pVFS6
P2 P3 P4 P5 P6 P7 P8 P9 P10 Pll P1 (partial) $2 S3a $5 and $6 $6
The plasmids pVFP2 through pVFS6 were identified by restriction analysis and blot hybridization (see results). The fragments are designated as assigned in Table 1 pVFP5 and pVFS3a and the other between p V F P l a and pVFS2. To our knowledge only one other complete chloroplast DNA library, that of Vigna radiata, has been reported to date (Palmer and Thompson 1981a). Other attempts to clone entire chloroplast genomes have yielded only partial libraries (Palmer and Thompson 1981a; Rochaix 1977).
Chloroplast DNA Mapping The order of the DNA fragments was determined by combining information from double digestion of native
chloroplast DNA, double digestion of the 15 cloned fragments and blot hybridization of the clones to complete digestions of native chloroplast DNA. To determine the relationship of the overlapping Kpnl, Xhol and Pstl fragments, the cloned fragments were used as 32p labelled probes against Pstl, Kpnl and Xhol digests of native chloroplast DNA bound on nitrocellulose filters. Pstl was also a positive control for the Pstl and Pstl-EcoR1 cloned fragments. An additional Sall digest was used as a positive control for Sall clones. A HindIII digest of lambda DNA served as the negative control. The blot hybridization results confirmed the authenticity of the clones. The hybridization results are shown in Fig. 5 and the data summarized in Table 3. Plasmids containing the Pstl fragments were digested with Pstl-Kpnl and Pstl-Xhol enzyme pairs. The Pstl-EcoR1 cloned fragment was similarly digested. The Sall clones were analyzed with three enzyme pairs: Sall-Pstl, Sall-Kpnl and Sall-Xhol. The electrophoretic pattern of double digestion P4, P9 and P10 are shown in Fig. 6 and the data from all double digestion experiments are tabulated in Table 4 (Figures for the remaining clones have not been presented to save space). The Pstl cloned chloroplast fragments, retrieved by Pstl digestion, were subsequently digested with Kpnl or Xhol. The double digestion results help to determine, more precisely, the location of the Kpnl or Xhol sites within the Pstl fragment. The P6, P7, P8, P10 and P l l fragments were untouched by Kpnl indicating that they were completely internal fragments of Kpnl fragments. Similarly, the Pstl fragments P8, P4, P10 and P11 were internal fragments of Xhol f r a g m e n t s . P4 and P9 were cut once; P3 and P5 were cut twice and P2 was cut three times by Kpnl. Xhol cuts the P6
Table 3. Summary of blot hybridization results 32p Labelled probe
Pstl
Kpnl
Xhol
Sall
pVFP2 pVFP3 pVFP4 pVFP5 pVFP6 pVFP7 pVFP8 pVFP9 pVFP10 pVFP11 pVFPla pVFS2 pVFS3a pVFS5/6
P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P1 P1 P1, P5 P1
K4, K7b, K10, K11 K2, K3, K12 K3, K9 K1, K5, K7a K2 K2 K5 K5, K9 K2 K5 K6, K7b, K8 K1, K6, K8 K1, K7a K1
X2, X3, X10b X4, X8, X10a, X12 X1, X4, X9 X1, X6, Xll X7, X8 X3, X7 X1 X1 X3 X1 X2, X5 X2, X5 X1, X6, Xll X5, X6
$2 S3a $5, $6
The plasmids pVFP2 through pVFS5/6 were used as 32p labelled probes against nitrocellulose bound Pstl, Kpnl, Xhol, Sail fragments. The fragments that resulted in hybridization with the probe are indicated as assigned in Table 1. Results for pVFP2 through pVFPll are shown in Fig. 5.
260
K. Ko et al.: Mapping the Chloroplast DNA of Viciafaba
Fig. 5. Blot hybridization of 32p-labelled Vicia faba clones to Pstl, Xhol and Kpnl digest of chloroplast DNA. The cloned probes used were pVFP2 through pVFPll in sequential order as indicated by lanes 1 through 10. The data are summarized in Table 3.
and P7 once; P2, P4 and P5 twice and P3 three times. Complete Kpnl and Xhol fragments within Pstl fragments were found to migrate with a fragment found in both single and double digest of native chloroplast DNA. The Pstl-EcoR1 fragment P1 a, was similarly analyzed by double digestion. Kpnl cuts the fragment twice and Xhol cuts it once. One of the fragments (K6) produced in the Pstl-Kpnl digest was completely within the Pstl-EcoR1 fragment. The other Kpn fragments were
overlapping P l a was initially identified by the presence o f the expected 1.9 kb fragment o f K7b produced in a Pstl-Kpnl double digest of Pla. The observation that P l a hybridizes to K7 confirms that this 1.9 kb fragment comes from K7 (data compiled in Table 3, figure not shown). The cloned Sall fragments, after Sall digestion, were subsequently digested with three enzymes: Pstl, Kpnl and Xhol. The Sall fragments, $2, $5 and $6 were untouched by Pstl, whereas the S3a was cut once.
K. Ko et al.: Mapping the Chloroplast DNA of Viciafaba
261 f6s
Fig. 6. Double digestion patterns of three selected Pstl clones. Clones pVFP4, pVFP9 and pVFP10 are digested with Pstl, Pstl-Kpn and Pstl-Xhol. The clones are digested in the above order for pVFP4 (lanes 2-4), pVFP9 (lanes 5-7) and pVFP10 (lanes 8-10). HindIlI digested lambda DNA is in lane 1. Data from the double digestions are summarized in Table 4
Kpnl did n o t c u t $5 and $6, b u t it did cut S3a once and $2 twice. X h o l did n o t cut $6 but it did c u t Sail fragments $2 and S5 once and S3a twice. The positions o f the Sall fragments were d e t e r m i n e d b y b l o t hybridizat i o n o f the nick translated Sall clones to chloroplast D N A digested w i t h Pstl, Kpnl and Xhol as well as Sall (data summarized in Table 3, Figure n o t shown). The data f r o m b o t h double digestion and blot hybridization yield the restriction m a p for Vicia faba as s h o w n in Fig. 7. The basic order o f the Kpnl fragments is in a g r e e m e n t w i t h the map c o n s t r u c t e d b y Koller and
Fig. 7. Physical map of the Viciafaba chloroplast chromosome. The map shows the restriction sites for Pstl, Xhol and Kpnl. The positions of three genes are also shown. The orientation of the LS gene is indicated. The fragments indicated inside the map (marked Pla, $2, $6, $5 and S3a) are the fragments cloned to complete the clone bank
Delius (1980) using d e n a t u r a t i o n m a p p i n g ; h o w e v e r , o u r m a p contains t w o additional fragments. The existence o f these fragments has been r e p o r t e d in an e v o l u t i o n a r y s t u d y b y Palmer and T h o m p s o n (1982). The position
Table 4. Data for double digestion of clones Cloned fragments
Pstl
Kpnl
pVFP2 pVFP3 pVFP4 pVFP5 pVFP6 pVFP7 pVFP8 pVFP9 pVFP10 pVFP11 pVFPla pVFS2 pVFS3a pVFS5/6 pVFS6
20.0 15.5 14.5 13.0 10.0 6.3 5.2 1.8 1.1 0.6 18.0 16.5 8.2, 4.5 8.4, 1.9 1.9
12.5, 3.1, 2.7, 1.1 8.0, 6.8, 0.6 12.0, 2.5 5.7, 4.5, 3.7 10.0 6.3 5.2 1.0, 0.8 1.1 0.6 8.6 (2x), 1.9 6.6, 6.0, 4.3 9.6, 3.3 8.4, 1.9 1.9
Xhol 9.7, 7.0, 7.4, 3.6, 8.6, 3.3, 9.7, 2.3, 9.4, 0.6 5.2, 0.8 5.2 1.8 1.1 0.6 13.0, 5.6 8.8 (2x) 7.3, 4.2, 5.5, 2.2, 1.9
Sail 2.9 3.0, 0.6 2.8 1.3
1.3 1.9
16.5 12.5 8.4 1.9
Each cloned fragment was retrieved by either Pstl or Sall digest. Kpnl or Xhol represents the fragments produced as a result of the double digestion (see text). In the case of Sall clones, an additional double digest with Pstl is shown, pVFS5/6 contain both $5 and $6. Results of $6 double digest are also shown, therefore double digestion of $5 can be resolved. All fragment sizes are given in kilobase pairs. Plasmid fragments are not recorded
K. Ko et al.: Mapping the Chloroplast DNA of Vieiafaba
262
Fig. 8. Blot hybridization of 32p end labelled E. eoli 16S and 23S rRNAs to Vicia faba chloroplast DNA fragments. The rRNAs are hybridized to Pstl and Xhol chloroplast fragments. The position of the bands are indicated by the arrows. The data are summarized in Table 5
Table. 5. Summary of gene mapping hybridizations Probe
Pstl
Xhol
Kpnl
Sall
16S rRNA 23S rRNA LS gene
P3 P3 P8, P9
X8 X8, X10a X1
K5
Sla
32p labelled rRNAs and pJZA4 were hybridized to nitrocelRltbse bound Pstl, Xhol and/or Kpnl and Sall fragments of Vicia faba chloroplast DNA
Fig. 9. Blot hybridization of a cloned spinach LS sequence (pJZA4) to Pstl, Xhol, Sall and Kpnl Vicia faba fragments. The data is summarized in Table 5
of our Sall cloned fragments in P1 is also consistent with the arrangement constructed by Koller and Delius (1981).
Chloroplast Gene Mapping Ribosomal R N A Genes. Chloroplast ribosomal RNA genes were detected with end labelled E. coli 16S and
23S ribosomal RNAs. The rRNA probes were hybridized to nitrocellulose bound PsH and X h o l fragments of chloroplast DNA. The hybridization results are presented in Fig. 8 and the data summarized in Table 5. Both 16S and 23S E. coli ribosomal RNAs hybridized to P3 fragment indicating that both rRNAs were contained within this fragment. The 16S rRNA hybridized to the X8 fragment. The 23S rRNA hybridized to both the X8 and X10 fragments. The positions of the rRNAs are shown on the map in Fig. 7. The RNA genes were mapped within a limit of 7,000 base pairs (bp).
Ribulose-l,5-Bisphosphate Carboxylase Large Subunit Gene. The location of the large subunit (LS) gene was determined by using a cloned BamHI-Aval fragment (pJZA4) encoding the LS gene in spinach, as the probe (Erion et al. 1981). The cloned fragment was 2,200 base pairs in length. The 32p labelled spinach fragment was hybridized to Pstl, Xhol, Sall and K p n l fragments of chloroplast DNA. The results are shown in Fig. 9 and summarized iri Table 5. pJZA4 hybridized to the X1 fragment, K5 fragment, one of the 31.0 kb Sall fragment, P8 and P9. The position of the LS gene for Vicia faba is indicated on the map in Fig. 7. The Vieia faba LS gene was located within a 3,000 bp limit. The polarity of the Viciafaba LS gene was determined by reversing the hybridization experiment between pJZA4 and pVFPg. The direction of transcription in the spinach LS gene has been determined by Erion et al., (1981). Transcription proceeds from the Aval end to the BamHI end. Several restriction sites have also been mapped within the BamHI-Aval fragment. A HindlII site is located approximately 700 bp from the BamHI site, a K p n l site at approximately 850 bp and a Pstl site at 1,500 bp from the BamHI site. After linearizing withBamHI, pJZA4 was subsequently digested with one of the above enzymes. The gel was blotted and hybridized with 32p labelled pVFP9. Hybridization did not occur with the BamHI-HindlII fragment of pJZA4. This suggests the portion of the Vicia faba LS gene encoded in pVFP9 (the 0.8 kb Pstl-Kpnl fragment) was not homologous to the BamHI-HindlII fragment of pJZA4. The results appear to indicate that the portion of the LS gene encoded in pVFP9 was located at the beginning of the gene. The polarity of the LS gene is indicated on the map in Fig. 7. Figure 7 combines data from this study and that of Koller and Delius to present a map of V. faba with 4 restriction enzymes. The clones available in our defined library are the fragments P2-Pll and the clones of the Sal fragments 2, 3a, 5 and 6. The fragment that completes the library is a partial EcoR1 digest fragment of P1 cloned in pVFPla. We are currently studying the transcriptional and translational products of these cloned fragments.
K. Ko et al.: Mapping the Chloroplast DNA of Vieiafaba
Acknowledgements. This research was supported by a grant from the National Science and Engineering Research Council.
References Bedbrook JR, Bogorad L (1976) Proc Natl Acad Sci USA 73: 4309-4313 Bedbrook JR, Kolodner R (1979) Ann Rev Plant Physiol 30: 593-620 Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HW, Boyer HW, Crosa JH, Falkow S (1977) Gene 2:95-113 Bovenberg WA, Kool AJ, Nijkamp HJJ (1981) Nucleic Acids Res 9:503-517 Chu NM, Oishi KK, Tewari KK (1981) Plasmid 6:279-292 Chu NM, Tewari KK (1982) Mol Gen Genet 286:23-32 Cohen SN, Chang ACY, Hsu L (1972) Proc Natl Acad Sci USA 69:2110-2114 Davis RW, Botstein D, Roth JR (1980) Advanced Bacterial Genetics. Cold Spring Harbor, New York, pp 116-117 Dugaiczyk A, Boyer JW, Goodman HM (1975) J Mol Biol 96: 171-184 Erion JL, Tarnowski J, Weissbach H, Brot H (1981) Proc Natl Acad Sci USA 78:3459-3463 Fluhr R, Edelman M (1981) Mol Gen Genet 181:484-490 Gordon KHJ, Crouse EJ, Bohnert HJ, Herrmann RG (1981) Genetics 59:281-296 Herrmann RG, Whitfield PR, Bottomley W (1980) Gene 8: 179-191
263 Koller B, Defius H (1980) Mol Gen Genet 178:216-269 Link G, Chambers SE, Thompson JA, Falk H (1981) Mol Gen Genet 181:454-457 Maniatis T, Jeffery A, van de Sande H (1975) Biochem 14: 3787-3794 Manning JE, Wolstenholme DR, Ryan RS, Hunter JA, Richards OC (1971) Proc Natl Acad Sci USA 68:1169-1173 Maxam AM, Gilbert W (1980) In: Grossmann L, Moldave K (eds) Method in Enzymology. Academic Press, London, pp 499-560 Mural RJ, Chesney RH, Vapnek D, Kropf MM, Scott JR (1979) Virology 93:387-397 Palmer JD (1982) Gene 10:1593-1605 Palmer JD, Thompson WF (1981a) Gene 15:21-26 Palmer JD, Thompson WF (1981b) Proc Natl Acad Sci USA 78:5533-5537 Palmer JD, Thompson WF (1982) Cell 29:537-550 Rawson JRY, Clegg MT, Thomas K, Rinehart C, Wood B (1981) Gene 16:11-19 Rhodes PR, Kung SD (1981) Can J Biochem 59:911-915 Rochaix JD (1977) In: Bogorad L, Weil JH (eds) Nucleic Acids and Protein Synthesis in Plants. Plenum Press, New York, pp 177-183 Southern EM (1975) J Mol Biol 98:503-517 van Ee JH, Vos YJ, Planta RJ (1980) Gene 12:191-200
C o m m u n i c a t e d by C. W. Birky, Jr. Received December 1, 1982 / February 1, 1983
7
CUrrt © Springer-Verlag 1983
Mapping the Chloroplast DNA of Viciafaba Kenton Ko, Neil A. Straus, and John P. Williams Department of Botany, University of Toronto, Toronto, Ontario, Canada, M5S 1A1
Summary. A complete clone bank representing the chloroplast DNA from Vicia faba has been constructed. A total of 15 fragments (10 Pstl, 1 Pstl-EcoR1 and 4 Sall fragments) were inserted into the vector pBR322 and transformed into the E coli strain HB101. The cloned fragments were used as the main tools in constructing the physical map of Vicia faba for the restriction endonucleases Pstl, Kpnl and Xhol. The identity of the cloned fragments was demonstrated by restriction analysis and blot hybridization. The information generated was used to construct the map. The 16S and 23S rRNA genes and the gene for the large subunit ofribulose1,5-bisphosphate carboxylase have been positioned on the map using heterologous probes. The orientation of the gene for the large subunit of RuBP carboxylase has also been determined. Key words: Chloroplast DNA clone bank - Physical mapping - Gene mapping
Introduction The chloroplast genome of most vascular plants consists of a single circular molecule containing 130-150 kilobase pairs (kb) (Bedbrook and Kol0dner 1979). A 2 0 - 2 5 kbp sequence of DNA that includes the ribosomal RNA genes has been shown in a number of species to be repeated once and arranged in an inverted orientation. The presence of the inverted repeated region has been verified by restriction endonuclease mapping for Zea mays, corn (Bedbrook and Bogorad 1976), Spinacea oleracea, spinach (Herrmann et al. 1980), Spirodela
Offprint requests to: K. Ko
oligorrhiza (van Ee et al. 1980), Sinapsis alba, mustard, (Link et al. 1981), Petunia hybrida, petunia, (Bovenberg et al. 1981), Oenothera parviflora (Gordon et al. 1981), Nieotiana tabaeum, tobacco (Fluhr and Edelman 1981), Vigna radiata, mung bean (Palmer and Thompson 1981 b), Pennisetum americanum (Rawson et al. 1981), Atriplex triangularis (Palmer 1982) and Cucumis sativa, cucumber (Palmer 1982). The chloroplast chromosome of at least three species of the family Fabaceae (Leguminosae) differ from the regular pattern of sequence arrangement. Vicia faba, broad bean (Koller and Delius 1980), Pisum sativum, pea (Palmer and Thompson 1981; Chu et al. 1981), and L3cer arietum (Chu and Tewari 1982) have smaller genomes which lack the inverted region and contain only one copy of the genes for chloroplast ribosomal RNA (rRNA). In this study we have physically mapped the chloroplast chromosome of Vicia faba with three restriction endonucleases and created a complete gene library in 15 cloned fragments. Our map confirms the basic arrangement of a Kpnl map published earlier (KoUer and Delius 1980) but includes two fragments that were overlooked in the previous study. In addition we have positioned the genes for the large subunit of ribulose1,5-bisphosphate carboxylase and the 16S and 23S ribosomal RNA.
Materials and Methods
Chloroplast DNA Isolation. Chloroplast DNA was isolated from 4 week old Vieiafaba (L. cv. Broad Windsor) plants by a modification of the method of Manning et al. (1971). The plants were destarched by keeping them in the dark for 12-36 h prior to isolation. The leaves (150-200 g) were quickly frozen in liquid N2 and blended in a Waring blender by the method of Rhodes and Kung (1981). The liquid N2 was allowed to evaporate. When
256 the blended leaves reached - 5 °C, approximately 900 ml of ice cold isolation buffer A [0.33 M sorbitol, 10 mM Tris pH 8.0, 50 mM ethylenediaminetetraacetic acid (EDTA), 5 mM 2mercaptoethanol, 0.1% bovine serum albumin (BSA) (w/v)l was added. The extract was filtered through two layers of cheesecloth and one layer of nylon mesh (20 /~m pore size). The filtrate was centrifuged at 1,500 x g for 15 min at 4 °C. The pellet was resuspended in 50 ml of ice cold isolation buffer B [10% sucrose (w/v), 10 mM Tris pH 8.0, 50 mM EDTA, 5 mM 2-mercaptoethanol, 0.1% BSA (w/v)]. Then 120 ml of ice cold isolation buffer C [75% sucrose (w/v), 10 mM Tris pH 8.0, 50 mM EDTA, 5 mM 2-mercaptoethanol, 0.1% BSA (w/v)l was added to give a final sucrose concentration of 5 5 56%. The mixture was centrifuged at 16,000 x g for 50 rain at 4 ° C. The floating chloroplast layer was collected and resuspended in isolation buffer B. The suspension was centrifuged at 1,000 x g for 20 min at 4 °C and the resulting pellet was resuspended in 6 ml of 10 mM Tris pH 8.0, 10 mM EDTA. The chloroplasts were lysed by adding 750 td of 20% sodium lauryl sulphate (SLS). Then 400 #1 of 5 M NaC1 was added and the lysate was deproteinized by extracting with equal volumes of Tris buffered phenol, followed by CHC13:isoamyl alcohol [24: l(v/v)l. The DNA was precipitated by the addition of two volumes of cold ethanol and collected by spooling onto a glass rod. The DNA was redissolved in 10 mM Tris pH 8.0, 1 mM EDTA. RNAse A was added to a final concentration of 100/~g/ml and the mixture was incubated at 37 °C for 1 h. The solution was deproteinizedwith sequentialphenol and CHC13 extractions as described earlier. The DNA was reprecipitated and redissolved in distilled H20.
Plasmid Isolation. Plasmids were isolated by a modification of the method of Mural et al. (1979). Plasmid-containing bacteria were grown in M9 minimal medium to an optical density of 0.6 at 650 nm. Two ml of 100 mg/ml chloramphenicol was added to 1 1 of culture and the culture was incubated overnight at 37 °C. The cells were harvested by eentrifugation at 700 x g for 10 min and resuspended in 40 ml of 10% sucrose (w/v), 50 mM Tris pH 8.0 Sixteen ml of 0.2 M EDTA pH 8.0 and 8 ml of lysozyme (5 mg/ml) was added and the mixture kept on ice for 20 rain. The cells were gently lysed by adding 4 ml 2% Sarkosyl. The lysate was clarified by centrifugation for 1.5 h at 13,000 rpm in a HB-4 rotor at 4 °C. The supematant was deproteinized as described in the chloroplast DNA isolation. The DNA was ethanol precipitated and recovered by centrifugation at 12,000 x g for 20 rain at 4 °C. Plasmid DNA was further purified by centrifugation to equilibrium in cesium chlorideethidium bromide gradients as described by Davis et al. (1980).
Restriction Endonuclease
Analysis. Chloroplast DNA was digested with various restriction endonucleases according to the suppliers instructions (Bethesda Research Laboratories, Inc.; New England Biolabs Inc.). Digested DNA's were electrophoresed in 0.7% horizontally submerged agarose gel for 1 8 - 2 0 h at 1.5 volts per cm of gel. The electrophoresis buffer was 80 mM Tris, pH 8.0, 10 mM sodium acetate, 2 mM EDTA. The gels were stained with ethidium bromide and visualized on a UV transilluminator.
K. Ko et al.: Mapping the Chloroplast DNA of Vicia faba transformants and their plasmids isolated from small cultures were analyzed by restriction endonucleases and agarose gel electrophoresis. The authenticity of the recombinant plasmids containing chloroplast DNA fragments was confirmed by blot hybridization to total restricted chloroplast DNA.
Radioactive Labelling of Probes. The recombinant plasmids and pJZA4 were labelled with a32P dCTP (Amersham Corp.) according to the supplier's instructions in a nick translation kit purchased from Bethesda Research Laboratories Inc. The plasmid, pJZA4 was a generous gift from Dr. S. D. Kung. E. coli rRNA was end labelled with 732p ATP (Amersham Corp.) according to Maxam and Gilbert (1980). The E. coli rRNAs were a generous gift from G. P. Singh.
Blot Hybridization. DNA fragments were transferred onto nitrocellulose sheets (Schleicher and Schuell - BA85) according to Southern (1975). The nitrocellulose filters were hybridized with 32p probes according to Fluhr and Edelman (1981).
Results and Discussion Restriction Endonuclease Analysis C h l o r o p l a s t D N A i s o l a t e d f r o m Vicia faba was digested w i t h various r e s t r i c t i o n e n z y m e s . T h e e l e c t r o p h o r e t i c p a t t e r n s o f t h e digestion p r o d u c t s are p r e s e n t e d in Fig. 1. S o m e o f t h e e n z y m e s s u c h as B a m H I and HindlII, digested t h e c h l o r o p l a s t D N A i n t o a large n u m b e r o f f r a g m e n t s (Fig. 1 a n d 2) while t h e r e m a i n i n g e n z y m e s p r o d u c e d a m o d e r a t e n u m b e r o f f r a g m e n t s (Fig. 2). T h e r e s t r i c t i o n f r a g m e n t sizes p r o d u c e d b y e n z y m e s K p n l , PsH, Sall a n d X h o l are c o m p i l e d in T a b l e 1. T h e f r a g m e n t sizes were e s t i m a t e d using HindlII digested l a m b d a D N A f r a g m e n t s as m o l e c u l a r size m a r k e r s (Maniatis e t al., 1975). K p n l , Pstl a n d X h o l were s e l e c t e d as t h e p r i m a r y m a p p i n g e n z y m e s because t h e y p r o d u c e d a m o d e r a t e n u m b e r o f discrete f r a g m e n t s t h a t were resolvable b y gel e l e c t r o p h o r e s i s . C h l o r o p l a s t D N A was also a n a l y z e d w i t h t w o pairs o f r e s t r i c t i o n e n z y m e s : P s t l - K p n l and P s t l - X h o l . These d o u b l e digestion p a t t e r n s are s h o w n in Fig. 3 a n d t h e f r a g m e n t sizes are c o m p i l e d in T a b l e 1. B o t h d o u b l e digestions p r o d u c e d a set o f 2 4 discrete fragments. T h e t o t a l m o l e c u l a r size o f t h e c h l o r o p l a s t D N A was e s t i m a t e d b y a d d i n g t h e f r a g m e n t sizes in b o t h single a n d d o u b l e digestions. These results are c o m p u t e d as t o t a l s in T a b l e 1. T h e e s t i m a t e d average overall size was
DNA Cloning. Pstl, Sail and Pst-EcoR1 restriction fragments of chloroplast DNA were inserted into the appropriate sites of pBR322 with T 4 DNA ligase by the method of Dugaiczyk et al. (1975). The recombinant plasmids were transformed into the E. coli strain HB101 according to Cohen et al. (1972). Colonies containing plasmids with inserts of chloroplast DNA were detected by their appropriate antibiotic phenotypes. Selected
Fig. 1. Gel electrophoresis of restriction endonuclease digests • of Vicia faba chloroplast DNA. Restriction patterns are shown for Smal (lane 2), BamHI (lane 3), HindlI1 (lane 4), Pstl (lane 5), Sall (lane 6) and HindlfI digested lambda DNA (lanes 1 and 7). The fragment sizes for the Sall digest are compiled in Table 1. Fragment sizes are given in kilobase pairs
K. Ko et al.: Mapping the Chloroplast DNA of Vicia faba
257
Table 1. Restriction enzyme analysis of chloroplast DNA
Pst l 35.0 20.0 15.5 14.5 13.0 10.0 6.3 5.2 1.8 1.1 0.6
(P1) (P2) (P3) (P4) (P5) (P6) (P7) (P8) (P9) (P10) (Pll)
123,000
Xho l
Kpnl
Sail
20.5 (X1) 19.5 (X2) 16.0 (X3) 15.0 (X4) 14.0 (X5) 12.0 (X6) 10.5 (X7) 4.2 (X8) 3.2 (X9) 3.0 (X10a & b) 1.25 ( X l l ) 0.6 (X12)
24.5 23.5 20.0 12.5 10.5 8.1 5.0 4.3 4.1 3.0 1.1 0.6
122,650
122,200
(K1) (K2) (K3) (K4) (KS) (K6) (K7a & b) (K8) (K9) (K10) (Kll) (K12)
31.0 16.5 12.5 10.0 8.4 1.6
(Sla & b) ($2) (S3a & b) ($4) ($5) (S6)
123,800
Pstl-Kpnl
Pstl-Xho l
20.5 12.5 11.5 10.0 8.0 (2x) 7.0 6.2 5.2 4.8 4.5 4.3 3.6 3.1 (2x) 2.6 (2x) 1.8 1.0 (2x) 0.9 0.76 0.7 0.6
14.5 12.0 10.0 9.4 (3x) 8.1 7.2 (2x) 5.2 (2x) 3.5 3.2 3.0 (3x) 2.1 1.8 1.3 1.1 0.9 0.6 (3x)
124,260
122,300
Native chloroplast DNA was digested with the restriction endonucleases indicated. Fragment sizes were determined using HindIII digested lambda DNA as size markers. The fragments produced are given in kilobase pairs. The totals are recorded in base pairs. The brackets in Pstl, Xhol, Kpnl and Sall indicate fragment designations. The brackets in Pstl-Kpnl and Pstl-Xhol indicate the number of co-migrating bands
Fig. 2. Gel electrophoretic patterns of Vicia faba chloroplast DNA digested with the three mapping endonucleases. Restriction patterns are shown for Pstl (lane 2), Kpnl (lane 3) and Xhol (lane 4). HindIII digest of lambda DNA is the size markers in lane 1. Fragment sizes are given in kilobase pairs
258
K. Ko et al.: Mapping the Chloroplast DNA of Viciafaba Construction o f Chloroplast DNA Clone Bank
Fig. 3. Double digestion patterns of Vicia faba chloroplast DNA. The double digestion patterns are shown for Pstl-Xhol (lane 2), Pstl (lane 3), Pstl-Kpnl (lane 4) and Xhol (lape 5). HindlII digest of lambda DNA is in lane 1. Fragment sizes are given in kilobase pairs
123,000 base pairs. The size estimated in this study is comparable to the size reported by Koller and Delius (1980). They reported 79.8 x 106 daltons or approximately 120,000 base pairs. However, they have overlooked two K p n l fragments totalling 1,700 base pairs or approximately 1.1 x 106 daltons. With these two K p n l fragments, the total size should be 80.9 x 106 daltons or about 122,000 base pairs. Blot hybridization confirmed the presence of these fragments.
A clone bank representing the entire chloroplast genome from V. faba was constructed. This clone bank was used as a source of discrete restriction fragments for physical mapping. In addition, it is a source of chloroplast genes to study the molecular genetics of photosynthesis. Pstl was selected as the primary cloning enzyme due to the fragments produced. The fragments generated were all well resolved; none of the eleven fragments were comigrating. Ten out of eleven of the Pstl fragments were cloned into the single Pstl site within the ampicillin resistant gene of pBR322 (Bolivar et al. 1977). Figure 4 shows the electrophoretic profile of all ten of these clones after digestion with Pstl. Approximately 73% of the chloroplast DNA was cloned into the Pstl site of pBR322. However, the largest Pstl fragment (35 kb) was not recovered, probably because the fragment is simply too large for direct cloning. In order to bring the fragment to a workable size, the Pstl fragment was isolated on a 10-30% sucrose gradient and partially digested with EcoR1 in the hope of cutting the majority of the fragments in half. The partial fragments were inserted into pBR322 that had been digested with both Pstl and EcoR1. One third of the fragment was cloned by this strategy, increasing the portion of the chloroplast genome cloned to 83%. The cloned fragment was identified by double digestion and blot hybridization (see mapping results). In order to clone the remaining part of the chloroplast genome, a partial clone bank in pBR322 of Sall fragments of V. faba chloroplast DNA was created and those fragments that overlap the Pstl fragments were selected (see mapping section). The Sall fragments that overlap Pstl fragments correspond to the Sall fragments 3a, 2, 5 and 6 of Koller and Delius (1980). The Vicia faba chloroplast DNA clone bank is complete in fifteen cloned fragments (see Table 2). Two areas of overlap exist; one between the cloned fragment
Fig. 4. Gel electrophoretic analysis of the ten Pstl cloned fragments. Lanes 1 and 16 contain HindIII digested lambda DNA. Lanes 2 and 15 contain Pstl digested chloroplast DNA: Lanes 3 and 14 contain Pstl digested pBR322. Lanes 4 through 13 contain Pstl digested clones from pVFP2 through pVFP11 in sequential order
K. Ko et al.: Mapping the Chloroplast DNA of Viciafaba
259
Table 2. ViciaFaba chloroplast DNA clone bank pBR322 Clone designation
Fragment cloned
pVFP2 pVFP3 pVFP4 pVFP5 pVFP6 pVFP7 pVFP8 pVFP9 pVFP10 pVFP11 pVFPla pVFS2 pVFS3a pVFS5/6 pVFS6
P2 P3 P4 P5 P6 P7 P8 P9 P10 Pll P1 (partial) $2 S3a $5 and $6 $6
The plasmids pVFP2 through pVFS6 were identified by restriction analysis and blot hybridization (see results). The fragments are designated as assigned in Table 1 pVFP5 and pVFS3a and the other between p V F P l a and pVFS2. To our knowledge only one other complete chloroplast DNA library, that of Vigna radiata, has been reported to date (Palmer and Thompson 1981a). Other attempts to clone entire chloroplast genomes have yielded only partial libraries (Palmer and Thompson 1981a; Rochaix 1977).
Chloroplast DNA Mapping The order of the DNA fragments was determined by combining information from double digestion of native
chloroplast DNA, double digestion of the 15 cloned fragments and blot hybridization of the clones to complete digestions of native chloroplast DNA. To determine the relationship of the overlapping Kpnl, Xhol and Pstl fragments, the cloned fragments were used as 32p labelled probes against Pstl, Kpnl and Xhol digests of native chloroplast DNA bound on nitrocellulose filters. Pstl was also a positive control for the Pstl and Pstl-EcoR1 cloned fragments. An additional Sall digest was used as a positive control for Sall clones. A HindIII digest of lambda DNA served as the negative control. The blot hybridization results confirmed the authenticity of the clones. The hybridization results are shown in Fig. 5 and the data summarized in Table 3. Plasmids containing the Pstl fragments were digested with Pstl-Kpnl and Pstl-Xhol enzyme pairs. The Pstl-EcoR1 cloned fragment was similarly digested. The Sall clones were analyzed with three enzyme pairs: Sall-Pstl, Sall-Kpnl and Sall-Xhol. The electrophoretic pattern of double digestion P4, P9 and P10 are shown in Fig. 6 and the data from all double digestion experiments are tabulated in Table 4 (Figures for the remaining clones have not been presented to save space). The Pstl cloned chloroplast fragments, retrieved by Pstl digestion, were subsequently digested with Kpnl or Xhol. The double digestion results help to determine, more precisely, the location of the Kpnl or Xhol sites within the Pstl fragment. The P6, P7, P8, P10 and P l l fragments were untouched by Kpnl indicating that they were completely internal fragments of Kpnl fragments. Similarly, the Pstl fragments P8, P4, P10 and P11 were internal fragments of Xhol f r a g m e n t s . P4 and P9 were cut once; P3 and P5 were cut twice and P2 was cut three times by Kpnl. Xhol cuts the P6
Table 3. Summary of blot hybridization results 32p Labelled probe
Pstl
Kpnl
Xhol
Sall
pVFP2 pVFP3 pVFP4 pVFP5 pVFP6 pVFP7 pVFP8 pVFP9 pVFP10 pVFP11 pVFPla pVFS2 pVFS3a pVFS5/6
P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P1 P1 P1, P5 P1
K4, K7b, K10, K11 K2, K3, K12 K3, K9 K1, K5, K7a K2 K2 K5 K5, K9 K2 K5 K6, K7b, K8 K1, K6, K8 K1, K7a K1
X2, X3, X10b X4, X8, X10a, X12 X1, X4, X9 X1, X6, Xll X7, X8 X3, X7 X1 X1 X3 X1 X2, X5 X2, X5 X1, X6, Xll X5, X6
$2 S3a $5, $6
The plasmids pVFP2 through pVFS5/6 were used as 32p labelled probes against nitrocellulose bound Pstl, Kpnl, Xhol, Sail fragments. The fragments that resulted in hybridization with the probe are indicated as assigned in Table 1. Results for pVFP2 through pVFPll are shown in Fig. 5.
260
K. Ko et al.: Mapping the Chloroplast DNA of Viciafaba
Fig. 5. Blot hybridization of 32p-labelled Vicia faba clones to Pstl, Xhol and Kpnl digest of chloroplast DNA. The cloned probes used were pVFP2 through pVFPll in sequential order as indicated by lanes 1 through 10. The data are summarized in Table 3.
and P7 once; P2, P4 and P5 twice and P3 three times. Complete Kpnl and Xhol fragments within Pstl fragments were found to migrate with a fragment found in both single and double digest of native chloroplast DNA. The Pstl-EcoR1 fragment P1 a, was similarly analyzed by double digestion. Kpnl cuts the fragment twice and Xhol cuts it once. One of the fragments (K6) produced in the Pstl-Kpnl digest was completely within the Pstl-EcoR1 fragment. The other Kpn fragments were
overlapping P l a was initially identified by the presence o f the expected 1.9 kb fragment o f K7b produced in a Pstl-Kpnl double digest of Pla. The observation that P l a hybridizes to K7 confirms that this 1.9 kb fragment comes from K7 (data compiled in Table 3, figure not shown). The cloned Sall fragments, after Sall digestion, were subsequently digested with three enzymes: Pstl, Kpnl and Xhol. The Sall fragments, $2, $5 and $6 were untouched by Pstl, whereas the S3a was cut once.
K. Ko et al.: Mapping the Chloroplast DNA of Viciafaba
261 f6s
Fig. 6. Double digestion patterns of three selected Pstl clones. Clones pVFP4, pVFP9 and pVFP10 are digested with Pstl, Pstl-Kpn and Pstl-Xhol. The clones are digested in the above order for pVFP4 (lanes 2-4), pVFP9 (lanes 5-7) and pVFP10 (lanes 8-10). HindIlI digested lambda DNA is in lane 1. Data from the double digestions are summarized in Table 4
Kpnl did n o t c u t $5 and $6, b u t it did cut S3a once and $2 twice. X h o l did n o t cut $6 but it did c u t Sail fragments $2 and S5 once and S3a twice. The positions o f the Sall fragments were d e t e r m i n e d b y b l o t hybridizat i o n o f the nick translated Sall clones to chloroplast D N A digested w i t h Pstl, Kpnl and Xhol as well as Sall (data summarized in Table 3, Figure n o t shown). The data f r o m b o t h double digestion and blot hybridization yield the restriction m a p for Vicia faba as s h o w n in Fig. 7. The basic order o f the Kpnl fragments is in a g r e e m e n t w i t h the map c o n s t r u c t e d b y Koller and
Fig. 7. Physical map of the Viciafaba chloroplast chromosome. The map shows the restriction sites for Pstl, Xhol and Kpnl. The positions of three genes are also shown. The orientation of the LS gene is indicated. The fragments indicated inside the map (marked Pla, $2, $6, $5 and S3a) are the fragments cloned to complete the clone bank
Delius (1980) using d e n a t u r a t i o n m a p p i n g ; h o w e v e r , o u r m a p contains t w o additional fragments. The existence o f these fragments has been r e p o r t e d in an e v o l u t i o n a r y s t u d y b y Palmer and T h o m p s o n (1982). The position
Table 4. Data for double digestion of clones Cloned fragments
Pstl
Kpnl
pVFP2 pVFP3 pVFP4 pVFP5 pVFP6 pVFP7 pVFP8 pVFP9 pVFP10 pVFP11 pVFPla pVFS2 pVFS3a pVFS5/6 pVFS6
20.0 15.5 14.5 13.0 10.0 6.3 5.2 1.8 1.1 0.6 18.0 16.5 8.2, 4.5 8.4, 1.9 1.9
12.5, 3.1, 2.7, 1.1 8.0, 6.8, 0.6 12.0, 2.5 5.7, 4.5, 3.7 10.0 6.3 5.2 1.0, 0.8 1.1 0.6 8.6 (2x), 1.9 6.6, 6.0, 4.3 9.6, 3.3 8.4, 1.9 1.9
Xhol 9.7, 7.0, 7.4, 3.6, 8.6, 3.3, 9.7, 2.3, 9.4, 0.6 5.2, 0.8 5.2 1.8 1.1 0.6 13.0, 5.6 8.8 (2x) 7.3, 4.2, 5.5, 2.2, 1.9
Sail 2.9 3.0, 0.6 2.8 1.3
1.3 1.9
16.5 12.5 8.4 1.9
Each cloned fragment was retrieved by either Pstl or Sall digest. Kpnl or Xhol represents the fragments produced as a result of the double digestion (see text). In the case of Sall clones, an additional double digest with Pstl is shown, pVFS5/6 contain both $5 and $6. Results of $6 double digest are also shown, therefore double digestion of $5 can be resolved. All fragment sizes are given in kilobase pairs. Plasmid fragments are not recorded
K. Ko et al.: Mapping the Chloroplast DNA of Vieiafaba
262
Fig. 8. Blot hybridization of 32p end labelled E. eoli 16S and 23S rRNAs to Vicia faba chloroplast DNA fragments. The rRNAs are hybridized to Pstl and Xhol chloroplast fragments. The position of the bands are indicated by the arrows. The data are summarized in Table 5
Table. 5. Summary of gene mapping hybridizations Probe
Pstl
Xhol
Kpnl
Sall
16S rRNA 23S rRNA LS gene
P3 P3 P8, P9
X8 X8, X10a X1
K5
Sla
32p labelled rRNAs and pJZA4 were hybridized to nitrocelRltbse bound Pstl, Xhol and/or Kpnl and Sall fragments of Vicia faba chloroplast DNA
Fig. 9. Blot hybridization of a cloned spinach LS sequence (pJZA4) to Pstl, Xhol, Sall and Kpnl Vicia faba fragments. The data is summarized in Table 5
of our Sall cloned fragments in P1 is also consistent with the arrangement constructed by Koller and Delius (1981).
Chloroplast Gene Mapping Ribosomal R N A Genes. Chloroplast ribosomal RNA genes were detected with end labelled E. coli 16S and
23S ribosomal RNAs. The rRNA probes were hybridized to nitrocellulose bound PsH and X h o l fragments of chloroplast DNA. The hybridization results are presented in Fig. 8 and the data summarized in Table 5. Both 16S and 23S E. coli ribosomal RNAs hybridized to P3 fragment indicating that both rRNAs were contained within this fragment. The 16S rRNA hybridized to the X8 fragment. The 23S rRNA hybridized to both the X8 and X10 fragments. The positions of the rRNAs are shown on the map in Fig. 7. The RNA genes were mapped within a limit of 7,000 base pairs (bp).
Ribulose-l,5-Bisphosphate Carboxylase Large Subunit Gene. The location of the large subunit (LS) gene was determined by using a cloned BamHI-Aval fragment (pJZA4) encoding the LS gene in spinach, as the probe (Erion et al. 1981). The cloned fragment was 2,200 base pairs in length. The 32p labelled spinach fragment was hybridized to Pstl, Xhol, Sall and K p n l fragments of chloroplast DNA. The results are shown in Fig. 9 and summarized iri Table 5. pJZA4 hybridized to the X1 fragment, K5 fragment, one of the 31.0 kb Sall fragment, P8 and P9. The position of the LS gene for Vicia faba is indicated on the map in Fig. 7. The Vieia faba LS gene was located within a 3,000 bp limit. The polarity of the Viciafaba LS gene was determined by reversing the hybridization experiment between pJZA4 and pVFPg. The direction of transcription in the spinach LS gene has been determined by Erion et al., (1981). Transcription proceeds from the Aval end to the BamHI end. Several restriction sites have also been mapped within the BamHI-Aval fragment. A HindlII site is located approximately 700 bp from the BamHI site, a K p n l site at approximately 850 bp and a Pstl site at 1,500 bp from the BamHI site. After linearizing withBamHI, pJZA4 was subsequently digested with one of the above enzymes. The gel was blotted and hybridized with 32p labelled pVFP9. Hybridization did not occur with the BamHI-HindlII fragment of pJZA4. This suggests the portion of the Vicia faba LS gene encoded in pVFP9 (the 0.8 kb Pstl-Kpnl fragment) was not homologous to the BamHI-HindlII fragment of pJZA4. The results appear to indicate that the portion of the LS gene encoded in pVFP9 was located at the beginning of the gene. The polarity of the LS gene is indicated on the map in Fig. 7. Figure 7 combines data from this study and that of Koller and Delius to present a map of V. faba with 4 restriction enzymes. The clones available in our defined library are the fragments P2-Pll and the clones of the Sal fragments 2, 3a, 5 and 6. The fragment that completes the library is a partial EcoR1 digest fragment of P1 cloned in pVFPla. We are currently studying the transcriptional and translational products of these cloned fragments.
K. Ko et al.: Mapping the Chloroplast DNA of Vieiafaba
Acknowledgements. This research was supported by a grant from the National Science and Engineering Research Council.
References Bedbrook JR, Bogorad L (1976) Proc Natl Acad Sci USA 73: 4309-4313 Bedbrook JR, Kolodner R (1979) Ann Rev Plant Physiol 30: 593-620 Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HW, Boyer HW, Crosa JH, Falkow S (1977) Gene 2:95-113 Bovenberg WA, Kool AJ, Nijkamp HJJ (1981) Nucleic Acids Res 9:503-517 Chu NM, Oishi KK, Tewari KK (1981) Plasmid 6:279-292 Chu NM, Tewari KK (1982) Mol Gen Genet 286:23-32 Cohen SN, Chang ACY, Hsu L (1972) Proc Natl Acad Sci USA 69:2110-2114 Davis RW, Botstein D, Roth JR (1980) Advanced Bacterial Genetics. Cold Spring Harbor, New York, pp 116-117 Dugaiczyk A, Boyer JW, Goodman HM (1975) J Mol Biol 96: 171-184 Erion JL, Tarnowski J, Weissbach H, Brot H (1981) Proc Natl Acad Sci USA 78:3459-3463 Fluhr R, Edelman M (1981) Mol Gen Genet 181:484-490 Gordon KHJ, Crouse EJ, Bohnert HJ, Herrmann RG (1981) Genetics 59:281-296 Herrmann RG, Whitfield PR, Bottomley W (1980) Gene 8: 179-191
263 Koller B, Defius H (1980) Mol Gen Genet 178:216-269 Link G, Chambers SE, Thompson JA, Falk H (1981) Mol Gen Genet 181:454-457 Maniatis T, Jeffery A, van de Sande H (1975) Biochem 14: 3787-3794 Manning JE, Wolstenholme DR, Ryan RS, Hunter JA, Richards OC (1971) Proc Natl Acad Sci USA 68:1169-1173 Maxam AM, Gilbert W (1980) In: Grossmann L, Moldave K (eds) Method in Enzymology. Academic Press, London, pp 499-560 Mural RJ, Chesney RH, Vapnek D, Kropf MM, Scott JR (1979) Virology 93:387-397 Palmer JD (1982) Gene 10:1593-1605 Palmer JD, Thompson WF (1981a) Gene 15:21-26 Palmer JD, Thompson WF (1981b) Proc Natl Acad Sci USA 78:5533-5537 Palmer JD, Thompson WF (1982) Cell 29:537-550 Rawson JRY, Clegg MT, Thomas K, Rinehart C, Wood B (1981) Gene 16:11-19 Rhodes PR, Kung SD (1981) Can J Biochem 59:911-915 Rochaix JD (1977) In: Bogorad L, Weil JH (eds) Nucleic Acids and Protein Synthesis in Plants. Plenum Press, New York, pp 177-183 Southern EM (1975) J Mol Biol 98:503-517 van Ee JH, Vos YJ, Planta RJ (1980) Gene 12:191-200
C o m m u n i c a t e d by C. W. Birky, Jr. Received December 1, 1982 / February 1, 1983
