- Email: [email protected]
[71] Restriction fragments from Chlamydomonas Chloroplast DNA
[71]
CHLOROPLAST D N A RESTRICTION FRAGMENTS
785
Acknowledgments This work was supported by grants from the National Institutes of Health (CA16006) and the American Cancer Society (NP2136) to Dr. J. E. Darnell. The author was a Fellow of the Jane CoffinChilds Memorial Fund for Medical Research. The author would also like to thank Dr. James E. Darnell for critically reading the manuscript.
[71] Restriction Fragments
from Chlarnydomonas
Chloroplast DNA By J. D. ROCHAIX
The chloroplast D N A of C h l a m y d o m o n a s reinhardii represents an attractive model system for studying the cooperation between the nucleocytoplasmic and organellar protein synthesizing apparatus in eukaryotic cells. This is largely due to the fact that this unicellular alga can be manipulated with ease both at the biochemical and genetic level. Since Sager's discovery of the first uniparental mutant in C. reinhardii, 1 numerous mutations of this type have been mapped, and there is now convincing evidence that these mutations are located in the chloroplast DNA. z'3 Electron microscopic 4 and restriction e n z y m e analyses 5'6 o f this D N A have shown that it is circular with a molecular weight of 125 × 106 to 130 x 106. A restriction map o f this D N A has been established recently. 7 The availability of pure restriction fragments o f the chloroplast D N A allows detailed studies on the organization and function o f this DNA. This article describes the methods used for obtaining chloroplast D N A restriction fragments, and some o f their properties. Chloroplast D N A preparation Media. TAP (Tris-acetate-phosphate) medium contains the same constituents as T M P medium 8 except that 1 ml o f glacial acetic acid is added per liter instead o f concentrated hydrochloric acid and the MgSO4 can be omitted.
1R. Sager, Proc. Natl. Acad. Sci. U.S.A. 40, 356 (1954). z R. Sager, "Cytoplasmic Genes and Organelles." Academic Press, New York, 1972. 3 N. W. Gillham, Annu. Rev. Genet. 8, 347 (1976). 4 W. Behn and R. G. Herrmann, Mol. Gen. Genet. 157, 25 (1977). 5 S. H. Howell, P. Heizmann, and S. Gelvin, in "Acides nucl6iques et synthbse de prot6ines chez les v6g6taux" (L. Bogorad and J. H. Weil, eds.), p. 313. CNRS, Paris, 1977. 6 j. D. Rochaix, in "Acides nucl6iques et synth~se de prot6ines chez les v6g6taux" (L. Bogorad and J. H. Weil, eds.), p. 77. CNRS, Paris, 1977. r j. D. Rochaix, J. Mol. Biol. 126, 597 (1978). S. J. Surzycki, this series, Vol. 23, p. 4.
METHODS IN ENZYMOLOGY, VOL. 65
Copyright © 1980 by Academic Press, lnc, All rights of reproduction in any form reserved~ 1SBN 0-12-181965-5
786
FUNCTIONAL SITE LOCALIZATION
[71]
Solutions
A Buffer: 0.1 M NaC1, 50 mM EDTA, 20 mM Tris-HC1, pH 8.0. Pronase solution: 10 mg/ml Pronase in 0.01 M Nacitrate, pH 5.0, predigested for 2 hr at 37° and stored frozen TE: 10 mM Tris-HC1, pH 8.0, 1 mM EDTA In order to obtain preparative amounts of high molecular weight chloroplast DNA, it is advisable to use the cell wall-deficient CWl5+ (Davies and PlaskitP) strain. This strain is grown on TAP medium at room temperature, and usually not more than 31 of culture are processed at a time. It is our experience that the yield and the quality of the chloroplast DNA decreases when large amounts of cells are processed together. When the cell density reaches 3 × 106 to 5 × 106 cells/ml, the cells are collected by low-speed centrifugation (4 min at 2500 g) in the cold. The pellet is resuspended gently with cold TAP medium, the cells are distributed equally into four 30-ml Sorvall Corex tubes, and centrifuged at 2000g for 5 min. Each of the four pellets is resuspended separately with 8 ml of cold A buffer. After gentle resuspension of the pellet, the cells are transferred into a 50-ml Erlenmeyer flask and 0.5 ml of Pronase solution is added. The cells are transferred into a 50° water bath after addition of 0.5 ml of 20% SDS. This procedure is repeated with the three other pellets. It is important not to pool the cell lysates before or during the incubation at 50° because difficulties with proper solubilization of the lysate may occur. Pronase solution is added after 45 min and again after 90 min of incubation. After 2 to 2.25 hr the color of the lysate starts turning from green to brown. At this point the lysates are cooled on ice, pooled together, and 2 volumes of distilled phenol (saturated with 0.1 M Na borate) are added. The mixture is shaken gently by hand, and after 20 min it is centrifuged in a Sorvall swingout rotor at 7000 g for 15 min. The aqueous phases are pooled and nucleic acids are precipitated with 2 volumes of EtoH. The DNA is spooled on a glass rod, rinsed in 70% EtoH, dried, and resuspended in TE buffer overnight at 4°. It is important to spool the DNA because centrifugation of the EtoH precipitate coprecipitates an unidentified component which will inhibit the restriction endonucleases later on. The resuspended nucleic acids are treated with 30 ~g/ml pancreatic RNase for 45 min at 37°. After phenol extraction the DNA is spooled, rinsed, dried, and resuspended in about 10 ml TE overnight at 4°. Four to six CsCI gradients are prepared by adding 3.35 ml of DNA solution to 4.27 g of solid CsC1. The gradients are centrifuged to equilibrium for at least 60 hr at 35,000 rpm in the Ti50 rotor at 18° to 20°. Gradients can be fractionated either with an ISCO fraction collector or 9 D. R. Davies and A. Plaskitt, Genet. Res. 17, 33 (1971).
[71]
CHLOROPLAST D N A RESTRICTION FRAGMENTS
a
b
R26 R25 R24, R24' R23 R22 R21 RI7
787
c
~ I3,Bgl4 12 II IO 9)
~
8
7)
RI3 RII ,RI2 RI0 R9 R8 R7
R6a ~R6b R5 R4 R3
6
m
5
4
R2 RI R01
[]
R02
R03 R04 R05 R07
FIG. 1. Agarose gel electrophoretic pattern of chloroplast DNA restriction fragments of C. reinhardii produced by EcoRI (a), B a m H I (b), and 8 g l l I (c). Electrophoresis was per-
formed in 0.8% agarose gels of 40 V for 14 hr at 4°.
a
b
c
d
e
R24
ROG
[71]
CHLOROPLAST D N A RESTRICTION FRAGMENTS
789
TABLE I SIZE OF CHLOROPLAST DNA RESTRICTION FRAGMENTSa Fragment no.
EcoRI
07 05 04 03 02 01 1 2 3 4 5 6a 6b 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24' 24 25 26
0.55R 0.72 0.76 0.81 0.85 0.93 1.16 1.25 1.76 1.90 1.97 2.14 2.14 2.23 2.30 2.50 2.78 3.10 3.10 3.20 3.70 3.70 3.83 4.10 4.54 4.60R 4.83 5.38R 5.55 6.35 6.65 6.70R 10.80 11.50
BamHI
BgllI
0.5 0.75 2.00R (3.40) 4.20 4.25R 4.66
0.80 1.10 1.50 1.60 2.55
5.20
2.96
6.00 (6.10) 7.10 8.10 10.60 12.30 13.90 16.20 23.75
(5.28) 6.20 (9.5) 11.95 16.75 18.5 27.8R 33.85R
a The EcoRI, BamHI and BglII fragments are indicated with their numbers in Fig. 1. Sizes are given in 10-e daltons. Fragments containing ribosomal RNA gene sequences are indicated by R. Fragments whose molecular weight is in parentheses are nonchloroplast DNA fragments (el. text).
FIG. 2. Agarose gel electrophoretic pattern of chloroplast DNA fragments obtained after digestion with SalI (a), HindIII (b), Sinai (c), PstI (d), and EcoRI (e).
790
FUNCTIONAL SITE LOCALIZATION
a
b
c
d
e
f
[71]
g
FIG. 3. Agarose gel electrophoretic patterns of chloroplast DNA plasmid hybrids digested w i t h B a m H I (slots a, b, d, e, f, g). Slot c represents a B a m H I chloroplast DNA digest. The arrow shows the linear pBR313 vector plasmid.
[71]
CHLOROPLAST D N A RESTRICTION FRAGMENTS
I 15
I I I
I0
I
I I I
m I
I
I I
I I
6
io
I I
I I I
I I
791
~o
I
I I
6'0
~'o
~6o
FlG. 4. (a) Heteroduplex formed between pCRI and a chloroplast plasmid hybrid containing Eco RI fragment R23. The thick arrow indicates the insertion site of the chloroplast DNA fragment into pCRI. The two thin arrows indicate the hairpin structures on the singlestranded chloroplast DNA which has a kinky appearance. Scale, 1 kb. (b) Drawing of 16 single-stranded R23 fragments with secondary structure features. manually by puncturing the b o t t o m o f the tube. The chloroplast D N A p e a k (8 = 1.696 g/ml) is readily visible on the light side of the nuclear D N A p e a k (8 = 1.724 g/ml). A second CsCI equilibrium density gradient is required in order to obtain pure chloroplast D N A with a final yield of 200 to 300/zg per 3 liters o f initial culture. Digestion of the Chloroplast D N A of Chlarnydomonas reinhardii with Restriction Endonucleases In order to achieve a c o m p l e t e digestion of the chloroplast D N A it is r e c o m m e n d e d to use two to three times the a m o u n t of restriction e n z y m e required to digest the same quantity of h D N A . Figure 1 shows an agarose gel pattern of chloroplast D N A fragments obtained after digesting the D N A with the restriction endonucleases E c o R I (a), B a m H I (b), and BglII (c). The fragments have been n u m b e r e d with increasing size from R07 to R26 for E c o R I , from B a l to B a l 5 for B a m H I , and from Bgl to B g l 4 for BglII. The size of these fragments has been m e a s u r e d by electron m i c r o s c o p y s and by c o m p a r a t i v e agarose gel electrophoresis using D N A fragments of known length as standards such as the h D N A E c o R I fragments 1° or the PM2 D N A H a e I I I fragments. 1~ The results are shown in Table I. The sum of the molecular weights of the 10M. Thomas and R. W. Davis, J. Mol. Biol. 91, 315 (1974). 1~K. S. Schmitz and B. R. Shaw, Science 197, 661 (1977).
792
FUNCTIONAL SITE LOCALIZATION
a
b
c
[71]
d
28K-
14K-
FIG. 5. Autoradiogram of in vitro synthesized polypeptides labeled with a3S-methionine and electrophoresed on a 12.5% SDS-polyacrylamide gel. The templates used are pCRI (slot a) and hybrid plasmids containingEcoRI fragment R15 (b), R12 (c), and R3 (d). (Courtesy of P. Malnoe.)
[71]
CHLOROPLAST DNA RESTRICTION FRAGMENTS
793
fragments produced by each of the three restriction enzymes is close to 126 × 106. It is apparent in Fig. 1 that several restriction fragments (whose numbers are in parentheses) are present in submolar amounts relative to the other chloroplast DNA fragments. We have shown that these fragments are contaminating nonchloroplast DNA fragments which originate from nuclear ribosomal DNA 12 (fragments Bal.1, Bal.2, Bg7) and from mitochondrial DNA 7 (Fragments Ba2, Ba8, Bg9; the corresponding EcoRI fragments comigrate with the fragments R18, R17, and R03). Figure 1 shows further that several restriction fragments are present in more than one molar amounts. Most of these fragments (i.e., fragments R07, R24, Bal, and Ba4) are repeated twice in the chloroplast genome and contain gene sequences of the chloroplast ribosomal RNA. 6,7 In contrast fragments R6a and R6b contain different" sequences, although they cannot be separated one from another under the conditions used. Further restriction endonuclease digestion patterns of the chloroplast DNA of C. reinhardii are shown in Fig. 2. Slots a, b, c, d, and e display SalI, HindIII, Sinai, PstI, and EcoRI chloroplast DNA fragments, respectively, fractionated by agarose gel electrophoresis. The size of the restriction fragments can be estimated from the known size of the EcoRI fragments (cf. Table I). Isolation of Chloroplast Restriction Fragments Chloroplast DNA restriction fragments can be prepared using two different approaches. Preparative Agarose Gel Electrophoresis. Several EcoRI fragments and most BamHI and BglII fragments can be obtained in pure form by cutting out the corresponding bands from preparative gels. The large chloroplast DNA fragments can be recovered efficiently from agarose gels by dissolving the agarose gel pieces with KI and by subsequent KI density centrifugation. ~a However, this approach is tedious. Molecular Cloning. Because several chloroplast DNA restriction fragments have almost the same size (e.g., fragments R18 and R19) it is not possible to purify them individually by agarose gel electrophoresis. These fragments can be inserted, however, in appropriate plasmids or bacteriophages. Over 80% of the chloroplast DNA of C. reinhardii has been cloned, 14 and most of the hybrid plasmids appear to be stable and faitht2 y. Marco and J. D. Rochaix, unpublished (1978). zaH. Blin, A. V. Gabain, and H. Bujard,FEBS Lett. 53, 84 (1975). 14j. D. Rochaix, in "Genetics and Biogenesis of Chloroplasts and Mitochondria" (T. Biicheret al., eds.), p. 375. Elsevier/North Holland, Amsterdam, 1976.
794
FUNCTIONAL SITE LOCALIZATION
[71]
fully replicated in the E. coli host. It is, therefore, possible to obtain large amounts of pure chloroplast DNA fragments. Figure 3 displays the pattern obtained when Bam chloroplast DNA hybrids are digested with BarnHI and electrophoresed on an agarose gel. The arrow indicates the linear vector plasmid pBR313, ~5 and the other fragments represent the cloned Bam restriction fragments which can be seen to correspond to the native B a m restriction fragments (slot c). Use of Chloroplast Restriction Fragments The availability of pure chloroplast DNA fragments, eithe~ purified by biochemical means or cloning, allows the study of several aspects of chloroplast DNA organization and function. a. Secondary structure of single-stranded chloroplast DNA. Figure 4 shows a heteroduplex formed between a hybrid plasmid containing E c o R I fragment R23 and the vector plasmid pCRI. Under these conditions only the chloroplast DNA appears to be single stranded. The thick arrow indicates the insertion site of the chloroplast DNA into the plasmid and the two thin arrows indicate two small hairpin structures, 100 to 200 nucleotides in length, which appear reproducibly in all the 16 molecules examined as shown in Fig. 4. These hairpin structures are also present in other chloroplast restriction fragments. b. Chloroplast restriction fragments allow the construction of a fine structure map of particular chloroplast genes. c. Use of chloroplast restriction fragments as templates for in vitro protein synthesis. Cloned chloroplas( DNA restriction fragments of C. reinhardii have been used as templates TM in the in vitro coupled transcription translation system of Zubay. 16 Figure 5 shows an autoradiogram of 35S-methionine-labeled in vitro synthesized polypeptides electrophoresed on an SDS:-polyacrylamide gel. It can be seen that the vector plasmid pCRI (slot a) produces two major polypeptides of 28,000 and 14,000 daltons in this system. Several of the chloroplast hybrids (slots b, c, and d) synthesize in addition polypeptides ranging in size between 10,000 and 60,000 daltons. It is possible to identify specific chloroplast genes contained in these hybrid molecules by immunoprecipitation of the in vitro products with specific chloroplast protein antisera. ~7 d. Chloroplast restriction fragments which contain known chloro15 F. Bolivar, R. L. Rodriguez, M. C. Betlach, and H. W. Boyer Gene 2, 75 (1977). 16 G. Zubay, D. A. Chambers, and L. C. Cheong, in "The Lactose Operon" (J. R. Beckwith and D. Zipser, eds.), p. 375. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1970. iT j. D. Rochaix and P. Malnoe, in "Chloroplast Development" (G. Akoyunoglou et al., eds.), p. 581. Elsevier/North Holland, Amsterdam, 1978.
[72]
TEMPLATE
FUNCTION
OF PHAGE
M13
REPLICATIVE
DNA
795
plast genes can be used for isolating specific chloroplast mRNAs, and they can be used as hybridization probes for measuring the concentration of individual mRNAs. e. Restriction enzyme analysis of the chloroplast DNA of uniparental mutants ofC. reinhardii 2,a should be particularly helpful forcorrelating the genetic and physical map of this chloroplast DNA. Acknowledgment This work was supported by the Swiss National Science Foundation no. 3.740.76.
[72] T e m p l a t e F u n c t i o n o f R e s t r i c t i o n E n z y m e F r a g m e n t s o f Phage M13 Replicative Form DNA B y R U U D N . H . KONINGS
I. I n t r o d u c t i o n
The F-specific filamentous coliphages, among which are included M 13, fl, fd, and ZJ/2, are unique among E. coli phages in that they do not kill or lyse their host cells (for a recent review, see Denhardt et al. 1). Their genome consists of a circular covalently closed single-stranded DNA molecule (MW about 2 × 106) which upon infection is converted into a double-stranded replicative form molecule (RF-I). Both in vivo as well as in vitro only the nonviral strand of this duplex DNA molecule functions as a template for transcription, z The DNA genome codes for at least nine polypeptides whose corresponding genes have been ordered into a circular genetic map. a,4 By using restriction enzyme fragments of replicative form DNA as a template, the technique of "DNA-dependent in vitro protein synthesis" has proved to be a very powerful tool both for the elucidation of the genetic organization as well as for the identification and localization of transcription-initiation (promoter-sites) and -termination signals on the D. T. Denhardt, D. Dressier, and D. S. Ray, "The Single Stranded DNA Phages." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1979. 2 R. N. H. Konings and J. G. G. Schoenmakers, in " T h e Single-Stranded DNA Phages" (D. T. Denhardt, D. Dressier, and D. S. Ray, eds.), p. 507-530. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1979. 3 C. A. van den Hondel, A. Weijers, R. N. H. Konings, and J. G. G. Schoenmakers, Eur. J. Biochem. 53, 559-567 (1975). 4 R. N. H. Konings, T. Hulsebos, and C. A. Van den Hondel,J. Virol. 15, 570-584 (1975).
METHODS IN ENZYMOLOGY, VOL. 65
Copyright ~) 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181965-5
CHLOROPLAST D N A RESTRICTION FRAGMENTS
785
Acknowledgments This work was supported by grants from the National Institutes of Health (CA16006) and the American Cancer Society (NP2136) to Dr. J. E. Darnell. The author was a Fellow of the Jane CoffinChilds Memorial Fund for Medical Research. The author would also like to thank Dr. James E. Darnell for critically reading the manuscript.
[71] Restriction Fragments
from Chlarnydomonas
Chloroplast DNA By J. D. ROCHAIX
The chloroplast D N A of C h l a m y d o m o n a s reinhardii represents an attractive model system for studying the cooperation between the nucleocytoplasmic and organellar protein synthesizing apparatus in eukaryotic cells. This is largely due to the fact that this unicellular alga can be manipulated with ease both at the biochemical and genetic level. Since Sager's discovery of the first uniparental mutant in C. reinhardii, 1 numerous mutations of this type have been mapped, and there is now convincing evidence that these mutations are located in the chloroplast DNA. z'3 Electron microscopic 4 and restriction e n z y m e analyses 5'6 o f this D N A have shown that it is circular with a molecular weight of 125 × 106 to 130 x 106. A restriction map o f this D N A has been established recently. 7 The availability of pure restriction fragments o f the chloroplast D N A allows detailed studies on the organization and function o f this DNA. This article describes the methods used for obtaining chloroplast D N A restriction fragments, and some o f their properties. Chloroplast D N A preparation Media. TAP (Tris-acetate-phosphate) medium contains the same constituents as T M P medium 8 except that 1 ml o f glacial acetic acid is added per liter instead o f concentrated hydrochloric acid and the MgSO4 can be omitted.
1R. Sager, Proc. Natl. Acad. Sci. U.S.A. 40, 356 (1954). z R. Sager, "Cytoplasmic Genes and Organelles." Academic Press, New York, 1972. 3 N. W. Gillham, Annu. Rev. Genet. 8, 347 (1976). 4 W. Behn and R. G. Herrmann, Mol. Gen. Genet. 157, 25 (1977). 5 S. H. Howell, P. Heizmann, and S. Gelvin, in "Acides nucl6iques et synthbse de prot6ines chez les v6g6taux" (L. Bogorad and J. H. Weil, eds.), p. 313. CNRS, Paris, 1977. 6 j. D. Rochaix, in "Acides nucl6iques et synth~se de prot6ines chez les v6g6taux" (L. Bogorad and J. H. Weil, eds.), p. 77. CNRS, Paris, 1977. r j. D. Rochaix, J. Mol. Biol. 126, 597 (1978). S. J. Surzycki, this series, Vol. 23, p. 4.
METHODS IN ENZYMOLOGY, VOL. 65
Copyright © 1980 by Academic Press, lnc, All rights of reproduction in any form reserved~ 1SBN 0-12-181965-5
786
FUNCTIONAL SITE LOCALIZATION
[71]
Solutions
A Buffer: 0.1 M NaC1, 50 mM EDTA, 20 mM Tris-HC1, pH 8.0. Pronase solution: 10 mg/ml Pronase in 0.01 M Nacitrate, pH 5.0, predigested for 2 hr at 37° and stored frozen TE: 10 mM Tris-HC1, pH 8.0, 1 mM EDTA In order to obtain preparative amounts of high molecular weight chloroplast DNA, it is advisable to use the cell wall-deficient CWl5+ (Davies and PlaskitP) strain. This strain is grown on TAP medium at room temperature, and usually not more than 31 of culture are processed at a time. It is our experience that the yield and the quality of the chloroplast DNA decreases when large amounts of cells are processed together. When the cell density reaches 3 × 106 to 5 × 106 cells/ml, the cells are collected by low-speed centrifugation (4 min at 2500 g) in the cold. The pellet is resuspended gently with cold TAP medium, the cells are distributed equally into four 30-ml Sorvall Corex tubes, and centrifuged at 2000g for 5 min. Each of the four pellets is resuspended separately with 8 ml of cold A buffer. After gentle resuspension of the pellet, the cells are transferred into a 50-ml Erlenmeyer flask and 0.5 ml of Pronase solution is added. The cells are transferred into a 50° water bath after addition of 0.5 ml of 20% SDS. This procedure is repeated with the three other pellets. It is important not to pool the cell lysates before or during the incubation at 50° because difficulties with proper solubilization of the lysate may occur. Pronase solution is added after 45 min and again after 90 min of incubation. After 2 to 2.25 hr the color of the lysate starts turning from green to brown. At this point the lysates are cooled on ice, pooled together, and 2 volumes of distilled phenol (saturated with 0.1 M Na borate) are added. The mixture is shaken gently by hand, and after 20 min it is centrifuged in a Sorvall swingout rotor at 7000 g for 15 min. The aqueous phases are pooled and nucleic acids are precipitated with 2 volumes of EtoH. The DNA is spooled on a glass rod, rinsed in 70% EtoH, dried, and resuspended in TE buffer overnight at 4°. It is important to spool the DNA because centrifugation of the EtoH precipitate coprecipitates an unidentified component which will inhibit the restriction endonucleases later on. The resuspended nucleic acids are treated with 30 ~g/ml pancreatic RNase for 45 min at 37°. After phenol extraction the DNA is spooled, rinsed, dried, and resuspended in about 10 ml TE overnight at 4°. Four to six CsCI gradients are prepared by adding 3.35 ml of DNA solution to 4.27 g of solid CsC1. The gradients are centrifuged to equilibrium for at least 60 hr at 35,000 rpm in the Ti50 rotor at 18° to 20°. Gradients can be fractionated either with an ISCO fraction collector or 9 D. R. Davies and A. Plaskitt, Genet. Res. 17, 33 (1971).
[71]
CHLOROPLAST D N A RESTRICTION FRAGMENTS
a
b
R26 R25 R24, R24' R23 R22 R21 RI7
787
c
~ I3,Bgl4 12 II IO 9)
~
8
7)
RI3 RII ,RI2 RI0 R9 R8 R7
R6a ~R6b R5 R4 R3
6
m
5
4
R2 RI R01
[]
R02
R03 R04 R05 R07
FIG. 1. Agarose gel electrophoretic pattern of chloroplast DNA restriction fragments of C. reinhardii produced by EcoRI (a), B a m H I (b), and 8 g l l I (c). Electrophoresis was per-
formed in 0.8% agarose gels of 40 V for 14 hr at 4°.
a
b
c
d
e
R24
ROG
[71]
CHLOROPLAST D N A RESTRICTION FRAGMENTS
789
TABLE I SIZE OF CHLOROPLAST DNA RESTRICTION FRAGMENTSa Fragment no.
EcoRI
07 05 04 03 02 01 1 2 3 4 5 6a 6b 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24' 24 25 26
0.55R 0.72 0.76 0.81 0.85 0.93 1.16 1.25 1.76 1.90 1.97 2.14 2.14 2.23 2.30 2.50 2.78 3.10 3.10 3.20 3.70 3.70 3.83 4.10 4.54 4.60R 4.83 5.38R 5.55 6.35 6.65 6.70R 10.80 11.50
BamHI
BgllI
0.5 0.75 2.00R (3.40) 4.20 4.25R 4.66
0.80 1.10 1.50 1.60 2.55
5.20
2.96
6.00 (6.10) 7.10 8.10 10.60 12.30 13.90 16.20 23.75
(5.28) 6.20 (9.5) 11.95 16.75 18.5 27.8R 33.85R
a The EcoRI, BamHI and BglII fragments are indicated with their numbers in Fig. 1. Sizes are given in 10-e daltons. Fragments containing ribosomal RNA gene sequences are indicated by R. Fragments whose molecular weight is in parentheses are nonchloroplast DNA fragments (el. text).
FIG. 2. Agarose gel electrophoretic pattern of chloroplast DNA fragments obtained after digestion with SalI (a), HindIII (b), Sinai (c), PstI (d), and EcoRI (e).
790
FUNCTIONAL SITE LOCALIZATION
a
b
c
d
e
f
[71]
g
FIG. 3. Agarose gel electrophoretic patterns of chloroplast DNA plasmid hybrids digested w i t h B a m H I (slots a, b, d, e, f, g). Slot c represents a B a m H I chloroplast DNA digest. The arrow shows the linear pBR313 vector plasmid.
[71]
CHLOROPLAST D N A RESTRICTION FRAGMENTS
I 15
I I I
I0
I
I I I
m I
I
I I
I I
6
io
I I
I I I
I I
791
~o
I
I I
6'0
~'o
~6o
FlG. 4. (a) Heteroduplex formed between pCRI and a chloroplast plasmid hybrid containing Eco RI fragment R23. The thick arrow indicates the insertion site of the chloroplast DNA fragment into pCRI. The two thin arrows indicate the hairpin structures on the singlestranded chloroplast DNA which has a kinky appearance. Scale, 1 kb. (b) Drawing of 16 single-stranded R23 fragments with secondary structure features. manually by puncturing the b o t t o m o f the tube. The chloroplast D N A p e a k (8 = 1.696 g/ml) is readily visible on the light side of the nuclear D N A p e a k (8 = 1.724 g/ml). A second CsCI equilibrium density gradient is required in order to obtain pure chloroplast D N A with a final yield of 200 to 300/zg per 3 liters o f initial culture. Digestion of the Chloroplast D N A of Chlarnydomonas reinhardii with Restriction Endonucleases In order to achieve a c o m p l e t e digestion of the chloroplast D N A it is r e c o m m e n d e d to use two to three times the a m o u n t of restriction e n z y m e required to digest the same quantity of h D N A . Figure 1 shows an agarose gel pattern of chloroplast D N A fragments obtained after digesting the D N A with the restriction endonucleases E c o R I (a), B a m H I (b), and BglII (c). The fragments have been n u m b e r e d with increasing size from R07 to R26 for E c o R I , from B a l to B a l 5 for B a m H I , and from Bgl to B g l 4 for BglII. The size of these fragments has been m e a s u r e d by electron m i c r o s c o p y s and by c o m p a r a t i v e agarose gel electrophoresis using D N A fragments of known length as standards such as the h D N A E c o R I fragments 1° or the PM2 D N A H a e I I I fragments. 1~ The results are shown in Table I. The sum of the molecular weights of the 10M. Thomas and R. W. Davis, J. Mol. Biol. 91, 315 (1974). 1~K. S. Schmitz and B. R. Shaw, Science 197, 661 (1977).
792
FUNCTIONAL SITE LOCALIZATION
a
b
c
[71]
d
28K-
14K-
FIG. 5. Autoradiogram of in vitro synthesized polypeptides labeled with a3S-methionine and electrophoresed on a 12.5% SDS-polyacrylamide gel. The templates used are pCRI (slot a) and hybrid plasmids containingEcoRI fragment R15 (b), R12 (c), and R3 (d). (Courtesy of P. Malnoe.)
[71]
CHLOROPLAST DNA RESTRICTION FRAGMENTS
793
fragments produced by each of the three restriction enzymes is close to 126 × 106. It is apparent in Fig. 1 that several restriction fragments (whose numbers are in parentheses) are present in submolar amounts relative to the other chloroplast DNA fragments. We have shown that these fragments are contaminating nonchloroplast DNA fragments which originate from nuclear ribosomal DNA 12 (fragments Bal.1, Bal.2, Bg7) and from mitochondrial DNA 7 (Fragments Ba2, Ba8, Bg9; the corresponding EcoRI fragments comigrate with the fragments R18, R17, and R03). Figure 1 shows further that several restriction fragments are present in more than one molar amounts. Most of these fragments (i.e., fragments R07, R24, Bal, and Ba4) are repeated twice in the chloroplast genome and contain gene sequences of the chloroplast ribosomal RNA. 6,7 In contrast fragments R6a and R6b contain different" sequences, although they cannot be separated one from another under the conditions used. Further restriction endonuclease digestion patterns of the chloroplast DNA of C. reinhardii are shown in Fig. 2. Slots a, b, c, d, and e display SalI, HindIII, Sinai, PstI, and EcoRI chloroplast DNA fragments, respectively, fractionated by agarose gel electrophoresis. The size of the restriction fragments can be estimated from the known size of the EcoRI fragments (cf. Table I). Isolation of Chloroplast Restriction Fragments Chloroplast DNA restriction fragments can be prepared using two different approaches. Preparative Agarose Gel Electrophoresis. Several EcoRI fragments and most BamHI and BglII fragments can be obtained in pure form by cutting out the corresponding bands from preparative gels. The large chloroplast DNA fragments can be recovered efficiently from agarose gels by dissolving the agarose gel pieces with KI and by subsequent KI density centrifugation. ~a However, this approach is tedious. Molecular Cloning. Because several chloroplast DNA restriction fragments have almost the same size (e.g., fragments R18 and R19) it is not possible to purify them individually by agarose gel electrophoresis. These fragments can be inserted, however, in appropriate plasmids or bacteriophages. Over 80% of the chloroplast DNA of C. reinhardii has been cloned, 14 and most of the hybrid plasmids appear to be stable and faitht2 y. Marco and J. D. Rochaix, unpublished (1978). zaH. Blin, A. V. Gabain, and H. Bujard,FEBS Lett. 53, 84 (1975). 14j. D. Rochaix, in "Genetics and Biogenesis of Chloroplasts and Mitochondria" (T. Biicheret al., eds.), p. 375. Elsevier/North Holland, Amsterdam, 1976.
794
FUNCTIONAL SITE LOCALIZATION
[71]
fully replicated in the E. coli host. It is, therefore, possible to obtain large amounts of pure chloroplast DNA fragments. Figure 3 displays the pattern obtained when Bam chloroplast DNA hybrids are digested with BarnHI and electrophoresed on an agarose gel. The arrow indicates the linear vector plasmid pBR313, ~5 and the other fragments represent the cloned Bam restriction fragments which can be seen to correspond to the native B a m restriction fragments (slot c). Use of Chloroplast Restriction Fragments The availability of pure chloroplast DNA fragments, eithe~ purified by biochemical means or cloning, allows the study of several aspects of chloroplast DNA organization and function. a. Secondary structure of single-stranded chloroplast DNA. Figure 4 shows a heteroduplex formed between a hybrid plasmid containing E c o R I fragment R23 and the vector plasmid pCRI. Under these conditions only the chloroplast DNA appears to be single stranded. The thick arrow indicates the insertion site of the chloroplast DNA into the plasmid and the two thin arrows indicate two small hairpin structures, 100 to 200 nucleotides in length, which appear reproducibly in all the 16 molecules examined as shown in Fig. 4. These hairpin structures are also present in other chloroplast restriction fragments. b. Chloroplast restriction fragments allow the construction of a fine structure map of particular chloroplast genes. c. Use of chloroplast restriction fragments as templates for in vitro protein synthesis. Cloned chloroplas( DNA restriction fragments of C. reinhardii have been used as templates TM in the in vitro coupled transcription translation system of Zubay. 16 Figure 5 shows an autoradiogram of 35S-methionine-labeled in vitro synthesized polypeptides electrophoresed on an SDS:-polyacrylamide gel. It can be seen that the vector plasmid pCRI (slot a) produces two major polypeptides of 28,000 and 14,000 daltons in this system. Several of the chloroplast hybrids (slots b, c, and d) synthesize in addition polypeptides ranging in size between 10,000 and 60,000 daltons. It is possible to identify specific chloroplast genes contained in these hybrid molecules by immunoprecipitation of the in vitro products with specific chloroplast protein antisera. ~7 d. Chloroplast restriction fragments which contain known chloro15 F. Bolivar, R. L. Rodriguez, M. C. Betlach, and H. W. Boyer Gene 2, 75 (1977). 16 G. Zubay, D. A. Chambers, and L. C. Cheong, in "The Lactose Operon" (J. R. Beckwith and D. Zipser, eds.), p. 375. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1970. iT j. D. Rochaix and P. Malnoe, in "Chloroplast Development" (G. Akoyunoglou et al., eds.), p. 581. Elsevier/North Holland, Amsterdam, 1978.
[72]
TEMPLATE
FUNCTION
OF PHAGE
M13
REPLICATIVE
DNA
795
plast genes can be used for isolating specific chloroplast mRNAs, and they can be used as hybridization probes for measuring the concentration of individual mRNAs. e. Restriction enzyme analysis of the chloroplast DNA of uniparental mutants ofC. reinhardii 2,a should be particularly helpful forcorrelating the genetic and physical map of this chloroplast DNA. Acknowledgment This work was supported by the Swiss National Science Foundation no. 3.740.76.
[72] T e m p l a t e F u n c t i o n o f R e s t r i c t i o n E n z y m e F r a g m e n t s o f Phage M13 Replicative Form DNA B y R U U D N . H . KONINGS
I. I n t r o d u c t i o n
The F-specific filamentous coliphages, among which are included M 13, fl, fd, and ZJ/2, are unique among E. coli phages in that they do not kill or lyse their host cells (for a recent review, see Denhardt et al. 1). Their genome consists of a circular covalently closed single-stranded DNA molecule (MW about 2 × 106) which upon infection is converted into a double-stranded replicative form molecule (RF-I). Both in vivo as well as in vitro only the nonviral strand of this duplex DNA molecule functions as a template for transcription, z The DNA genome codes for at least nine polypeptides whose corresponding genes have been ordered into a circular genetic map. a,4 By using restriction enzyme fragments of replicative form DNA as a template, the technique of "DNA-dependent in vitro protein synthesis" has proved to be a very powerful tool both for the elucidation of the genetic organization as well as for the identification and localization of transcription-initiation (promoter-sites) and -termination signals on the D. T. Denhardt, D. Dressier, and D. S. Ray, "The Single Stranded DNA Phages." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1979. 2 R. N. H. Konings and J. G. G. Schoenmakers, in " T h e Single-Stranded DNA Phages" (D. T. Denhardt, D. Dressier, and D. S. Ray, eds.), p. 507-530. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1979. 3 C. A. van den Hondel, A. Weijers, R. N. H. Konings, and J. G. G. Schoenmakers, Eur. J. Biochem. 53, 559-567 (1975). 4 R. N. H. Konings, T. Hulsebos, and C. A. Van den Hondel,J. Virol. 15, 570-584 (1975).
METHODS IN ENZYMOLOGY, VOL. 65
Copyright ~) 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181965-5