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Isolation and molecular weight of circular chloroplast DNA from Euglena gracilis
285
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 97143
I S O L A T I O N AND 1V[OLECULAR W E I G H T OF C I R C U L A R C H L O R O P L A S T DNA FROM EUGLENA G R A C I L I S JERRY
E. M A N N I N G AND O L I V E R C. R I C H A R D S
Department o/ Riochemistry, University o/ Utah Colldgc o/ Medicine, Salt Lake City, Utah 84112
(~:.s..4 .)
(Received O c t o b e r 25th, 197 I)
SUMMARY
I. Chloroplast DNA from Euglena gracilis has been examined by electron microscopy and by sedimentation analysis in order to establish its molecular size and in order to determine the feasibility of physical isolation of whole molecules. 2. Using bacteriophage 2 DNA as an internal standard, a molecular weight of 9.2 • lO 7 is obtained for chloroplast DNA by comparison of contour lengths of circular 2 and circular chloroplast DNA molecules found on identical grids in the electron microscope. Chloroplast DNA molecules were also found in a tightly twisted configuration; this is the first demonstration of supercoiled DNA structures in chloroplast preparations. 3. Sucrose gradient sedimentation analysis of chloroplast DNA with molecular wholes and halves of bacteriophage T 7 DNA gives a molecular weight for chloroplast DNA identical to that obtained b y electron microscopy. Furthermore, molecular whole and half nmlecules of chloroplast DNA can be effectively isolated by sucrose gradient sedimentation.
INTRODUCTION
The first demonstration of circular chloroplast DNA molecules was recently reported for DNA from chloroplasts of Euglena gracilis 1. The chloroplast origin of these DNA molecules was confirmed by the absence of contaminating organisms in the chloroplast fractions, by the characteristic buoyant density of the DNA present in the chloroplast fractions, and b y the absence of circular molecules in lysates of mitochondria from strains of E. gracilis. Studies of the kinetic complexity of this chloroplast DNA have placed a value of 1.8. lO s daltons on the genetic content of this DNA 2. If corrections are applied to these data to account for the low G + C content of this DNA 3, a genetic complexity of 9 " I°7 daltons is obtained. This value closely approaches our value for the molecular size of chloroplast DNA obtained by contour length measurements of circular DNA molecules in the electron microscope. In our previous report 1, a mass per unit length was assumed, utilizing the data of LANG4. This paper extends our previous results and defines the size of the chloroplast DNA both b y sedimentation techniques and by electron microscopic examination Biochim. Biophys. Acta, 259 (1972) 285-296
280
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MANNING, O. C. RICHARI)S
of purified chloroplast DNA in the presence of an internal standard, circular bacteriophage 2 DNA. A method for the preparation of substantial amounts of chloroplast DNA of full length is described.
MATERIALS AND METHODS
Culture conditions Euglena gracilis Klebs, strain Z, cells were maintained autotrophically, as described previously 5, in the presence of o.7o mC of i32Plphosphoric acid per 1 of medium and harvested in a Sorvall KSB-I continuous-flow centrifuge at 4 °. The cells were suspended in cold sucrose Tris-EDTA buffer (o.37 M sucrose, o.oi M Tris-HCl, o.o 5 M EDTA, pH 7.6) and centrifuged at 3ooo × g for IO rain. The pellet was suspended in 4 vol. of cold sucrose-Tris-EDTA buffer and used immediately for chloroplast isolation.
Isolation o/chloroplasts Euglena chloroplasts were isolated by modification of the procedure of el al. 1. After the second sucrose flotation, the floating pad of chloroplasts was washed 3 times in sucroseTris-EDTA buffer and centrifuged at 30o0 × g for IO rain. These chloroplasts were suspended in o.i M NaC1, o.o1 M Tris-HC1, o.oi M EDTA, pH 7.6, and centrifuged at IOOO× g for 5 rain. This washing was repeated twice, and the final chloroplast pellet was suspended in o.79 M sucrose, o.15 M NaC1, o.io M EDTA, 0.o 5 M TrisHC1, pH 9.o. The suspension was centrifuged at 7o00 × g for IO rain at 4 °. The pellet was suspended in I volume of the above sucrose solution per volume of packed chloroplasts and stored at --20 ° in aliquots of 2 ml. BRAWERMAN AND EISENSTADT6, as described by MANNING
Isolation o] DNA (a) Chloroplast DNA ]or buoyant density analysis in CsCl. A thawed suspension (4 ml) of chloroplasts was diluted with 4 ml of o.79 M sucrose, o.15 M NaCI, o.Io M EDTA, o.o 5 M Tris-HC1, pH 9.o. Sodium dodecyl sulfate, pronase, and sodium deoxyeholate were added to final concentrations of o.5 o.,o, I mg/ml, and I o/.,o,respectively, and the mixture was stirred overnight at 4 °. An equal volmne of buffer-saturated (o.15 M NaC1, o.io M EDTA, o.o 5 M Tris-HC1, pH 9.o), distilled phenol was added, and the mixture was shaken at 5-min intervals for 3o rain. The mixture was centrifuged at io o o o x g for IO rain at 4 °, and the aqueous layer was removed. The phenol layer was extracted with o. 5 volume of fresh buffer and centrifuged at IO ooo ×g for IO rain. The aqueous layer was removed and added to the previous aqueous layer. NaC104 (5 M) was added to I M final concentration, and the mixture was shaken at 5-min intervals over a 3o-min period with an equal volume of chloroform-isoamyl alcohol (24 : I, v/v). The mixture was centrifuged at IO ooo x g for IO rain at 4 °, and the aqueous layer was removed and dialyzed against 5 1 of o.I5 M NaC1, o.o15 M sodium citrate, o.5 mM EDTA, pH 8.o, overnight at 4 °. The solution was heated at 37 ° and incubated for 3o min with pretreate& pancreatic ribonuclease A (5°/~g/ml) and ribonuclease T 1 (5o units/ml). An equal volume of chloroform-isoainyl alcohol was added to the solution, and the mixture was shaken t3iochim. Biophys. ,4cla, 259 (1972) 285 296
287
MOLECULAR WEIGHT OF CHLOROPLAST D N A
at 5-rain intervals over a 3o-min period. The mixture was centrifuged at xo ooo ×g for IO rain at 4 °, and the aqueous layer was removed and dialyzed overnight as above and used for buoyant density analysis in CsC1.
(b) Chloroplast DNA /or electron microscopic examination and sucrose gradient analysis. A suspension of 2 ml of E. gracilis chloroplasts was thawed at 4 °. Sodimn dodecyl sulfate, pronase pretreated by the method of YOUNG AND SINSHEIMER s, and sodium deoxycholate were added to final concentrations of 0.5 o,,jo, I mg/ml, and 1 % , respectively, and the mixture was gently rocked for 5-rain periods at approximately I-h intervals for 12 h at 4 °. NaC10 4 (5 M) was added to I 3I final concentration, and the solution was rocked for 3o rain at 4 ° with an equal volume of buffer-saturated (o.15 M NaCl, o.io M EDTA, o.o5 M Tris-HC1, p H 9.o), distilled phenol and o. 5 volume of chloroform-isoamyl alcohol (24 : I, v/v). The mixture was centrifuged at IOOO × g for 15 min at 4 °. The aqueous phase was removed with a 2-ram bore Pasteur pipette and dialyzed overnight against o.15 M NaC1, O.Ol5 M sodium citrate, o. 5 mM EDTA, p H 8.o, in the cold and then prepared directly for electron microscopy or sucrose gradient analysis. (c) Bacteriophage DNA. 3H-labeled, T 7 DNA was isolated from T 7 bacteriophage grown in the presence of i3H]thymidine 9. Isolated all-labeled T 7 DNA was found to consist of a mixture of whole and half molecules by sedimentation analysis as described by STUDIER 10 a n d BRUNER AND VINOGRAD ll. DNA was isolated from 2 sus O29Cls57 bacteriophage by the methods described by THOMAS AND ABELSON9. DNA was then circularized by the procedure of MACI-IATTIE AND THOMAS ~2.
(d) Other DNA preparations. Escherichia coli bromouracil-labeled hybrid DNA, Microeoccus lysodeikticus DNA, and E. gracilis total cellular DNA were prepared as described previously 5. Electron microscopy Approximately IO/,1 of isolated DNA was added to o.I ml of I M ammonium acetate containing 0.05 °/o cytochrome c and 0.5 % formaldehyde. This solution was spread onto a hypophase of 0. 3 M ammonium acetate containing 0. 5 % formaldehyde or of water with 0.5 % formaldehyde. Preparation of grids, electron microscopy, and contour-length measurements were performed as described previously m3.
Sucrose gradient sedimentation A o.146 to o.585 M linear sucrose gradient (11. 5 ml) containing o.I M NaC1, I mM EDTA, and 0.05 M potassium phosphate, p H 6. 7, was layered into a polyallomer tube at 4 °. A DNA sample containing 0.50 ml of 32p-labeled chloroplast DNA and o.o15 ml of nil-labeled T 7 DNA was layered on top of the gradient and sedimented for 2.5 h at 4 ° ooo rev./min in a Spinco SW 41 rotor at 4 °. Fractions containing 6 drops each were collected from the bottom of the tube through an i8-gauge needle by allowing drops to run gently down the side of the collecting tubes. Gradients were analyzed by electron microscopy and for radioactivity.
CsCl equilibrium density gradient centri/ugation A solution (0.02 M Tris-HC1, p H 8.0) of 3.0 ml was prepared containing Biochim. Biophys. Ac/a, 259 (I972) 285-296
2~8
J.E.
MANNING, O. C. RICHARDS
E. gracilis a2p-labeled chloroplast DNA and E. coli bromouracil-labeled hybrid DNA (O = z.754 g/cm a) and non-radioactive chloroplast I)NA (p = 1.685 g/cm :~) as density markers. CsC1 (3.85 g) was added, and the tube was filled with mineral oil and centrifuged in the Spinco 4o.2 fixed-angle rotor at 35 ooo rev./min for 3 days at 2 5 . Fractions of 4 drops each were collected and diluted with o. 5 ml of o.I 5 M NaCI, o.o15 M sodium citrate, and their absorbance at 2(5o nm and radioactivity were determined. Analytical CsC1 equilibrium density gradient centrifugation was performed as described previously a. Scintillation counting Gradient fractions were prepared for radioactivity measurements bv two methods: (a) Fractions were diluted with o. 5 nfl of o.I 5 M NaC1, o.oI 5 M sodium citrate, 0.05 ml of sahnon sperm DNA (zoo #g), and 0.055 ml of 5.0 M KOH and were incubated at 37 ° for I6 h and then cooled at 4 ~. Cold o.61 M trichloroacetic acid (L43 ml) was added to a final concentration of 0. 3 M. The nfixture was kept on ice for 45 to 6o rain, 2 ml of cold water was added, and the precipitate was collected by vacumn filtration on 24-nun nitrocellulose filters, Type 136 (Selfleicher and Schuell). Filters were washed with two 3-nil portions of cold water, transferred to counting vials, and dried at 80'. For radioisotope determination, Io ml of toluene liquifluor (4.og of 2,5-bis-2-(5-tert.-tmtyl-benzoxazolyl)thiophene to 1 1 of toluene) was added to each vial. (b) Gradient fractions were treated with deoxyribonuclease 1 and snake venom phosphodiesterase by the method of I~.ELI.YANI) SM1TH14. O.50 ml of o.15 M NaC1, O.Ol5 M sodium citrate, 0.o5 ml of sahnon sperm DNA (zoo#g), and o.55 ml of o.6z M trichloroacetic acid were added, and the precipitate was collected and counted as in Method (a).
Reagents Beef pancreatic ribonuclease A, pancreatic deoxyribonuelease I, ribonuclease "1"1, and snake venom phosphodiesterase were obtained from Worthington Biochemical Corp. Salmon sperm DNA and pronase (B grade) were obtained from Calbiochem. Dried M. lysodeikticus cells were purchased from Sigma Chemical Co. Optical grade CsC1 was purchased from Harshaw Chemical Co. Carrier-free [a2P]phosphoric acid was purchased from New England Nuclear Corp.
RESULTS
Examination o] isolated chloroplast DNA in the electron microscope The linear density and molecular weight of circular chloroplast DNA molecules were accurately determined by use of circular bacteriophage 2 DNA as an internal control, a2P-labeled DNA was isolated from chloroplast fractions by gentle lysis with sodium dodecyl sulfate, sodium deoxyeholate, and pronase, followed by deproteinization with a chloroform-phenol mixture. This DNA was added to a solution containing circular ). DNA inolecules, and the mixture was prepared for examination in the electron microscope. Thus, both DNA's were prepared for microscopy under identical conditions. An average contour length of 44.5 (±0.6) #m was obtained for the circular chloroplast DNA and an average contour length of 14. 9 Biochim. Biophys..4cta, 259 (I972) 285 290
DNA
MOLECULAR WEIGHT OF CHLOROPLAST
289
(:ko.3) # m was found for the circular 2 DNA molecules, using 0.3 M ammonium acetate containing 0.5 % formaldehyde as the hypophase in the protein monolayer technique 15 (Fig. I). A picture of a circular chloroplast DNA molecule is shown in Fig. 2. Additionally, rare highly twisted circular molecules were observed (Fig. 3). The contour lengths of the twisted circles, although difficult to determine accurately, were not dissimilar from those obtained for the open circular molecules.
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Fig. ~. F r e q u e n c y distributions of the c o n t o u r lengths of circular D*~A molecules from isolated chloroplast DN"A and from 2 DNA. A m i x t u r e of chloroplast D N A and ). D N A was p r e p a r e d directly for electron microscopy, using a h y p o p h a s e of o. 3 }I a m m o n i u m acetate, o.5 °o forinaldehyde. The circular chloroplast D N A had an average c o n t o u r length of 44.5(±o.6)1,m and is s h o w n hy the cross-hatched area. Circular ~, D N A had a c o n t o u r length of I 4 . 9 ( - - o . 3 ) / m L illustrated by the area b o u n d e d b y h e a v y lines.
32P-labeled DNA from the same preparation of chloroplast DNA was spread with circular ), DNA onto a hypophase of water containing o. 5 °o formaldehyde. The DNA's were picked up on carbon-coated formvar grids and examined in the electron microscope. An average contour length of 48.9 ( ~ i . 2 ) / , m was obtained for the circular chloroplast DNA and an average contour length of 16.2 ( ~ o . 4 ) # m ,,,as found for the circular 2 DNA molecules. Thus, identical ratios of 3.0 for the the contour lengths of circular chloroplast DNA to circular 2 DNA were obtained for both conditions of spreading DNA. Comparison of the length measurements of the two circular DNA's allowed the best calculation of the molecular weight of the chloroplast DNA. The upper densitometer tracing in Fig. 4 shows the distribution of DNA extracted from whole cells of E. gracilis, strain Z, and shows minor peaks with buoyant densities of 1.69o and 1.685 g/cm ~, representative of mitochondrial and chloroplast DNA, respectively6'~6'tL DNA isolated from the same 3~P-labeled chloroplast fractions used for electron microscopy was banded in a CsC1 equilibrium density gradient. This DNA formed a band with a main peak at 1.685 g/cm 3 (Fig. 4, lower tracing), showing that the predominant DNA species is chloroplast DNA. A minor component Biochi~n. Biophys..dora, 259 (I972) 2 8 5 296
290
J. F.. MANNING, O. C. RICHARDS
Fig. 2. Electron micrograph of a rotary shadowed circular I_)•A molecule isolated from a chloroplast fraction from cells of Et~gh'na gracilis, strain Z. The molecule is 44 ,ran ill contour length; the line represents I/tin.
is o b s e r v e d w i t h a b u o y a n t d e n s i t y of 1.698 g / c m 3 ; t h i s D N A h a s b e e n s h o w n t o b e a p o r t i o n of c h l o r o p l a s t D N A e n r i c h e d in c i s t r o n s f o r c h l o r o p l a s t r i b o s o m a l R N A 18. This DNA has been observed previously, but the small quantity makes it unlikely that this DNA accounts for the circular molecules observed in our chloroplast pre-
tliochiln. 14iophyx, ..tcta, 259 (It~72) 285 296
MOLECULAR WEIGHT OF CHLOROPLAST
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Fig. 3. Electron m i c r o g r a p h of a r o t a r y shadowed, t i g h t l y twisted circular molecule of D N A isolated from a chloroplast fraction from cells of E. gracilis, strain Z. The line represents i ,urn.
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292
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MANNING, O. C. I(ICHARDS
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8u0y0ni density (g/cm5} Fig. 4- ] ) e n s i t o l n e t e r tracings of p h o t o g r a p h s after equilibriuln d e n s i t y g r a d i e n t c e n t r i f u g a t i o n i11 CsC1 of whole cell D N A (44 Hg, top tracing) a n d of isolated chloroplast D N A (-~ iig, b o t t o m tracinR). M. kvsodeikticus D N A (p 1.731 g/ell] 3) served as t h e reference I)NA.
p a r a t i o n s L l ; u r t h e r m o r e , MANNING t't al. 1 have shown t h a t tile circular molecules are not due to c o n t a m i n a t i o n b y l n i t o c h o n d r i a l D N A or b y other organisms. Thus, the circular D N A molecules are a t t r i b u t a b l e to chloroplast D N A .
Sucrose gradient sedimentation S e d i m e n t a t i o n of carefully isolated 32P-labeled D N A from chloroplasts t h r o u g h sucrose d e n s i t y g r a d i e n t s was used to s e p a r a t e classes of D N A molecules of different m o l e c u l a r weights. The s e d i m e n t a t i o n rate allowed a m e a s u r e m e n t of the size of chloroplast D N A i n d e p e n d e n t of t h a t o b t a i n e d by' electron microscopy. Molecular wholes a n d halves of a l l - l a b e l e d T 7 D N A were used as i n t e r n a l molecular weight m a r k e r s a n d allowed a c c u r a t e measure of the m o l e c u l a r weight of chloroplast D N A . A f t e r s e d i m e n t a t i o n t h e r a d i o a c t i v i t y of the different fractions was d e t e r m i n e d . Three distinct, r a p i d l y s e d i m e n t i n g a2p p e a k s were o b s e r v e d (Fig. 5); a s c a t t e r i n g of a'~p was found at the t o p of the t u b e a n d is not pictured. The relationship of distance t r a v e l e d in the g r a d i e n t to molecular weight, d e v e l o p e d b y BUR(;I AND HERSHEY 19 a n d modified b v I:REH:EI~II~:R~°, was used to d e t e r m i n e the molecular weights of m a t e r i a l in p e a k regions relative to T 7 D N A . Using a value of 2.52 • I o v for the molecular weight of the 3H-labeled T 7 D N A , a m o l e c u l a r weight of 9.2 • I o v was c a l c u l a t e d for the fastest sediinenting 3~p peak ( F r a c t i o n 3 i ) . Material in t h e second fastest sedim e n t i n g 32p p e a k (Fraction 40) corresponds to molecules h a v i n g an average molecular weight of 5.3 " IoL D N A i s o l a t e d from these '~"P-labeled chloroplast fractions was centrifuged in a p r e p a r a t i v e CsC1 equilibrium d e n s i t y g r a d i e n t with n o n - r a d i o a c t i v e chloroplast D N A a n d E. coli b r o m o u r a c i l - l a b e l e d h y b r i d D N A as b u o y a n t d e n s i t y m a r k e r s (l:ig. 0). The p e a k of :~zP-labeled D N A coincides with n o n - r a d i o a c t i v e m a r k e r chh)rophlst D N A from E. gracilis showing t h a t the a2p counts o b s e r v e d in tile sucrose g r a d i e n t ]¢iockim. H i o p k y s . . 4 c t a , -'50 (~(17") : , S 5 : 0 0
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Fig. 5. Sucrose d e n s i t y gradient s e d i m e n t a t i o n profile of 32P-labeled chloroplast DNA. w-'t'labeled D N A was gently isolated from purified chloroplasts of E. gracilis, strain Z, and layered on t o p of a o. i46 to o.585 M sucrose gradient with 3H-labeled T 7 DNA. Centrifugation was performed for 2. 5 tl at 4 ° ooo rev./min at 4 ~ in a SW 41 rotor in a Spinco Model L2-65 centrifuge. Drop fractions were collected and analyzed for radioactivity. 0 - - - O , a2p counts/rain; 0 - 0 , "H counts/ nlill.
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Fig. 6. Equilibrium d e n s i t y profile of a2P-labeled chloroplast D N A in CsCI d e n s i t y gradient. a2p-labeled D N A was isolated from purified chloroplasts of E. gracilis, strain Z, and nlixed with E. coil bromouracil-labeled h y b r i d D N A (p = 1.754 g/cnl s, n o n - r a d i o a c t i v e peak in F r a c t i o n s 18 to 25) and with chloroplast D N A (p -- 1.685 g/cm a, non-radioactive peak in Fractions 64 to 7 o) as d e n s i t y references. Centrifugation was performed in a neutral CsC1 density gradient at 35 ooo rev./min at 257 for 65 h in a Spinco 4o.2 fixed-angle rotor. Drop fractions were collected and their a b s o r b a n c e and r a d i o a c t i v i t y were determined. @ - - - O , 32p counts/min; 0 - 0 , absorbance at 260 nm, reference DNA; A - A , b o y a n t density. (Fig. 5) a r e p r e d o m i n a n t l y i n c h l o r o p l a s t D N A . F u r t h e r m o r e , t h e a c i d - p r e c i p i t a b l e , a l k a l i - s t a b l e r a d i o a c t i v i t y i n F r a c t i o n s 28 t h r o u g h 6 o o f t h e s u c r o s e g r a d i e n t ( F i g 5) can be completely removed by treatment with deoxyribonuclease and snake venom phosphodiesterase. Riochim. P, iophys..4cta, z59 (i972) 285 296
294
J.E.
MANNING, O. C. RICHARDS
Electron microscopy o~ peak/factions/rom the s~crose gradient Sedimentation of 32P-labeled chloroplast D N A was performed in a sucrose gradient using conditions identical to those of Fig. 5. Every third fraction was analyzed for radioactivity and a pattern similar to Fig. 5 was obtained. The two fractions on either side of the peak fractions were diluted with o.5o ml of o.I 5 M NaC1, o.oz5 M sodium citrate and combined. Preparations of DNA from the fastest sedimenting peak (Fig. 5, Vracti~ns 3o and 32) contained 3 ° ~i~ circular molecules which had an average contour length of 43.5 ( q ~.2)/~m (Fig. 7, area under dashed lines). I.inear molecules varied between 2 and 44 ktm (cross-hatched area) with 74 'I;, of all D N A molecules varying between 4 o and 45/~m. i8 0o of the DNA had contour lengths between z 4 and 27/tin with an average contour length of iq. 7/~m. 16
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Fi~. 7. F r e q u e n c y d i s t r i b u t i o n s of c i r c u l a r a n d l i n e a r I ) N , \ mol e c ul e s from t he f a s t e s t a n d second f a s t e s t s e d i m e n t i n g 321) p e a k s in t h e sucrose g r a d i e n t (Fig. 5), The l e n g t h d i s t r i b u t i o n ol l i n e a r D N A m o l e c u l e s fotmd in the f a s t e s t s e d i m e n t i n g ~2p p e a k is s h o w n b y t h e c r o s s - h a t c h e d area, a n d t h e l e n g t h d i s t r i b u t i o n for c i r c u l a r D N A m o l e c u l e s found in t h e s a m e p e a k is s h o w n b y t h e a r e a u n d e r t h e d a s h e d line. The a r e a b o u n d e d b y h e a v y lines s how s t h e l e n g t h d i s t r i b u t i o n of t h e l i n e a r D N A mo lecules found in the second f a s t e s t s e d i m e n t i n g 32p p e a k ; no c i r c u l a r D N A m olecules were o b s e r v e d in t h i s DNA. i " r e q u e n c y d i s t r i b u t i o n s are e x p r e s s e d as t h e p e r c e n t a g e of t h e t o t a l l e n g t h of ] ) N A e x a m i n e d in each peak. The t o t a l l e n g t h of D N A m e a s u r e d from t h e f a s t e s t s e d i m e n t i n g D N A w a s i 3 I 3 p m of w h i c h 3 ° °/o w a s in t h e form of circles. The t o t a l l e n g t h of I ) N A i n e a s u r e d from t h e second fa,stest s e d i m e n t i n g D N A w a s 95r/,,m.
Electron microscopic examination of the D N A molecules in the second fastest sedimenting peak (Fig. 5, Fractions 39 and 41) showed linear molecules having lengths between I and 3o/~m (Fig. 7, area bounded b y h e a v y lines) with 76.5 o~ of the DNA ranging in length from 14 to 27/ml. The D N A in this peak had an average contour length of 19. 7 #m. No circular molecules were observed.
l~i,;chim. Hiophys. _qcta, 250 (3972 ) 2~ 5 zq6
MOLECULAR WEIGHT OF CHLOROPLAST
DNA
295
DISCUSSION
The results indicate that at least one-third of the DNA from chloroplasts of
Euglena gracilis is in the form of circular molecules with an average contour length of 44.5 (~o.6)/~m under our conditions of electron microscopy. Covalently closed DNA molecules in the configuration of a supercoil were also observed; this is the first demonstration of DNA supercoils in chloroplast preparations. Since no linear molecules longer than the longest circle were observed, the DNA molecules in chloroplasts of E. gracilis in silu are (a) circular molecules of about 44/zm, or (b) hoth circular molecules of approximately 44/~m and linear molecules. At the present time it is not possible to favor one of the above two conclusions. The contour length of circular chloroplast DNA was accurately calibrated using circular 2 DNA for an internal standard on the same grids for the electron microscope. The average contour lengths for the circular 2 and circular chloroplast DNA were I4.9 # m and 44.5/,m, respectively, when the hypophase was o. 3 M amnmnium acetate containing o.5 °'o formaldehyde. Using 3.o6. IO7 for the molecular weight of 2 DNA 2°, chloroplast DNA has a molecular weight of 9.2 • IoL A mass per unit length of 2.o6 • IOn daltons per # m is obtained and compares well with that of 2.o 7 • IO6 daltons per/zm obtained by LANG~for duplex DNA examined under similar conditions. Stretching or contraction of DNA molecules caused by variation of the hypophase onto which the molecules are spread appears to be proportional to the length of the molecules. For example, whether the hypophase was water and o.5 °5 formaldehyde or o.3 M ammonium acetate and o.5 °'o formaldehyde, the same ratios of contour length of circular chloroplast DNA to circular 2 DNA were obtained. Thus, use of an internal standard in determination of the mass per unit length of DNA molecules is both essential and meaningful. Analysis of carefully isolated chloroplast DNA by sucrose gradient sedimentation showed that the DNA distinctly separated into three rapidly sedimenting fractions. The fastest fraction had an average molecular weight of 9.2 • lO7, as calculated from the sedimentation coefficient of 50 S 2°. This value for the molecular weight obtained by sedimentation techniques is the same as the molecular weight calculated from the average contour length of the molecules observed in the electron microscope. No increased frequency of circular molecules was observed in this fastest sedimenting peak; this m a y be a result of the increased manipulations involved in analyzing the sucrose gradient. Furthermore, no separation of circular and linear molecules of Minilar contour lengths was observed in the sucrose gradient, The average molecular weight of molecules in the second fastest peak was calculated from the sedimentation coefficient and was found to be 5.3 " ioL 76 °,o of the DNA in this peak had contour lengths between 14 and 27/~m, the lengths expected for shear products of whole molecules of chloroplast DNA broken into molecular halves. The molecular weight of chloroplast DNA using internal molecular weight standards for electron microscopy and for sedimentation analysis has been shown to be 9.2 " x& and revises the earlier estimate of 8.3 • xo7 (ref. x) based solely on electron microscopy. The accurate determination of the molecular weight of the chloroplast DNA molecule using two independent methods does, however, confirm our previous interpretation that multiple copies of this DNA molecule exist per chloroplast.
Biochim. Biophys. Xcta, 259 (1972) 285-296
296
j . F . . MANNING, o. C. RICHARDS
NOTE ADDED 1N PROOF: ( R e c e i v e d Cospreading that DNA
of 2 bacteriophage
o n e u n i t l e n g h t of 2 D N A
lenght.
Therefore,
February
less than
DNA
was mixed o.I
'~?, o f t h e
2nd, 1972 ) with chloroplast with
about
DNA
was performed
o n e c i r c l e of 4 4 - # m
circles observed
could
so
contour
be triple-lenght
molecules.
ACKNOWLEDGMENTS
This research was supported by Grant GB-2o763 from the National Science Foundation. One of us (J.E.M.) was supported by U.S. Public Health Service Training Grant GM-ooI52. We should like to thank I)r. D. R. \,Volstenhohne for use of his electron microscope and facilities. REFERENCES i J. E. MANNING, D. 14.. \\'OLSTI'2NHOLME, [{. ,~. RYAN, J, :\. HUNTER ANI) 1). C. RICHARDS, Proc. Natl. d c a d . Sci. U.S., 68 11971) II69. 2 E. STUTZ, FI'21IS I.ett., S (~97 o) 25. 3 J. (k WETMUR AND N, 1)AVIDSO.X, .f. ),lol. Biol., 31 (I06~) 3413. 4 D. LANG, f . Mol. Biol., 54 (~97 °) 557. 5 ()- C. I(ICHARDS, H. S. I(YAN AND J. E. ~[ANNIN(;, t3i,chim, l~iophys..qcta, 23 s (I97~) ~0o. 0 G. ~3RAWF.RMXN AND J. M. EIS~NSTAI)T, Hiochim. l¢i@hys, dcta. 0~ 119643 4777 J. MARMt:R, ,[. i~Icd. 13iol., 3 (1901 ) 2o8. S E . T. YOUNG AND 1{. 1.. SINSHEIIMGR, .l- d~'/0/, l]iol., 3 o (10(37) l05. 9 C. A. THOMAS AND J. ABELSON, ill (;. l.. CANTONI ANI) ]). 1(. I)AVIES, Procedures i~ Nucleic .4cid lfcsearck, H a r p e r a n d Row, Nexx York, 1960, p, 553io 1:. \V. STUDIO:R, .[. Mol. I~iol., 11 11005) 373. t l R . I~RUNER AND J. \rlNOGRAI), lliochim. Biophys..qcta, toS (1065) i8. 12 1.. A. MAcH.¢TTIE AND C. A. "I'Ho.~IAS, Scie~wC, i44 [t004) ,142. 13 1). 1<. \¥OLST~NltOL.XI~; AND N. J. (;ROSS, Proc. N a t l . . 4 c a d . Sci. U..q., ol (,gO,S) 245. 14 T . J. KELLY AND H. (). SMIT,L J. Mol. Biol., 5i (I97 °) 393. 15 ;\. K. KLI~:INSCHMIDT aND R. K. ZAHN, Z. Natur/orsch., 14b (1059) 77 ° • 10 D . S. RAY AXD 1'. C. HANAWALT, .1. 310l. 13iol., 1~ (I965) 76(3. 17 M. EDEL.XL.XN, J. A. St'HIFF AND H. T. EPSTI~IN, ,J. ]fol. Biol., i~ (,0o5) 700 . 18 E. STUTZ ANI) J. P. VA~DREY, F.I'2tLq Lell., 17 ([97 ~) 277. t0 E. BURGI AND ;\. D. [{F,RSIII*:}', t3iopkys. ,[., 3 ([903) 3o0 • 2o D. I:REIIq~:LDt,'R, .1. 3Ioi. l]iol., 54 (I97 o) 507 . 13iochim. ]~iopk3's..qcla, 259 (I072) 2~ 5 20()
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 97143
I S O L A T I O N AND 1V[OLECULAR W E I G H T OF C I R C U L A R C H L O R O P L A S T DNA FROM EUGLENA G R A C I L I S JERRY
E. M A N N I N G AND O L I V E R C. R I C H A R D S
Department o/ Riochemistry, University o/ Utah Colldgc o/ Medicine, Salt Lake City, Utah 84112
(~:.s..4 .)
(Received O c t o b e r 25th, 197 I)
SUMMARY
I. Chloroplast DNA from Euglena gracilis has been examined by electron microscopy and by sedimentation analysis in order to establish its molecular size and in order to determine the feasibility of physical isolation of whole molecules. 2. Using bacteriophage 2 DNA as an internal standard, a molecular weight of 9.2 • lO 7 is obtained for chloroplast DNA by comparison of contour lengths of circular 2 and circular chloroplast DNA molecules found on identical grids in the electron microscope. Chloroplast DNA molecules were also found in a tightly twisted configuration; this is the first demonstration of supercoiled DNA structures in chloroplast preparations. 3. Sucrose gradient sedimentation analysis of chloroplast DNA with molecular wholes and halves of bacteriophage T 7 DNA gives a molecular weight for chloroplast DNA identical to that obtained b y electron microscopy. Furthermore, molecular whole and half nmlecules of chloroplast DNA can be effectively isolated by sucrose gradient sedimentation.
INTRODUCTION
The first demonstration of circular chloroplast DNA molecules was recently reported for DNA from chloroplasts of Euglena gracilis 1. The chloroplast origin of these DNA molecules was confirmed by the absence of contaminating organisms in the chloroplast fractions, by the characteristic buoyant density of the DNA present in the chloroplast fractions, and b y the absence of circular molecules in lysates of mitochondria from strains of E. gracilis. Studies of the kinetic complexity of this chloroplast DNA have placed a value of 1.8. lO s daltons on the genetic content of this DNA 2. If corrections are applied to these data to account for the low G + C content of this DNA 3, a genetic complexity of 9 " I°7 daltons is obtained. This value closely approaches our value for the molecular size of chloroplast DNA obtained by contour length measurements of circular DNA molecules in the electron microscope. In our previous report 1, a mass per unit length was assumed, utilizing the data of LANG4. This paper extends our previous results and defines the size of the chloroplast DNA both b y sedimentation techniques and by electron microscopic examination Biochim. Biophys. Acta, 259 (1972) 285-296
280
J.E.
MANNING, O. C. RICHARI)S
of purified chloroplast DNA in the presence of an internal standard, circular bacteriophage 2 DNA. A method for the preparation of substantial amounts of chloroplast DNA of full length is described.
MATERIALS AND METHODS
Culture conditions Euglena gracilis Klebs, strain Z, cells were maintained autotrophically, as described previously 5, in the presence of o.7o mC of i32Plphosphoric acid per 1 of medium and harvested in a Sorvall KSB-I continuous-flow centrifuge at 4 °. The cells were suspended in cold sucrose Tris-EDTA buffer (o.37 M sucrose, o.oi M Tris-HCl, o.o 5 M EDTA, pH 7.6) and centrifuged at 3ooo × g for IO rain. The pellet was suspended in 4 vol. of cold sucrose-Tris-EDTA buffer and used immediately for chloroplast isolation.
Isolation o/chloroplasts Euglena chloroplasts were isolated by modification of the procedure of el al. 1. After the second sucrose flotation, the floating pad of chloroplasts was washed 3 times in sucroseTris-EDTA buffer and centrifuged at 30o0 × g for IO rain. These chloroplasts were suspended in o.i M NaC1, o.o1 M Tris-HC1, o.oi M EDTA, pH 7.6, and centrifuged at IOOO× g for 5 rain. This washing was repeated twice, and the final chloroplast pellet was suspended in o.79 M sucrose, o.15 M NaC1, o.io M EDTA, 0.o 5 M TrisHC1, pH 9.o. The suspension was centrifuged at 7o00 × g for IO rain at 4 °. The pellet was suspended in I volume of the above sucrose solution per volume of packed chloroplasts and stored at --20 ° in aliquots of 2 ml. BRAWERMAN AND EISENSTADT6, as described by MANNING
Isolation o] DNA (a) Chloroplast DNA ]or buoyant density analysis in CsCl. A thawed suspension (4 ml) of chloroplasts was diluted with 4 ml of o.79 M sucrose, o.15 M NaCI, o.Io M EDTA, o.o 5 M Tris-HC1, pH 9.o. Sodium dodecyl sulfate, pronase, and sodium deoxyeholate were added to final concentrations of o.5 o.,o, I mg/ml, and I o/.,o,respectively, and the mixture was stirred overnight at 4 °. An equal volmne of buffer-saturated (o.15 M NaC1, o.io M EDTA, o.o 5 M Tris-HC1, pH 9.o), distilled phenol was added, and the mixture was shaken at 5-min intervals for 3o rain. The mixture was centrifuged at io o o o x g for IO rain at 4 °, and the aqueous layer was removed. The phenol layer was extracted with o. 5 volume of fresh buffer and centrifuged at IO ooo ×g for IO rain. The aqueous layer was removed and added to the previous aqueous layer. NaC104 (5 M) was added to I M final concentration, and the mixture was shaken at 5-min intervals over a 3o-min period with an equal volume of chloroform-isoamyl alcohol (24 : I, v/v). The mixture was centrifuged at IO ooo x g for IO rain at 4 °, and the aqueous layer was removed and dialyzed against 5 1 of o.I5 M NaC1, o.o15 M sodium citrate, o.5 mM EDTA, pH 8.o, overnight at 4 °. The solution was heated at 37 ° and incubated for 3o min with pretreate& pancreatic ribonuclease A (5°/~g/ml) and ribonuclease T 1 (5o units/ml). An equal volume of chloroform-isoainyl alcohol was added to the solution, and the mixture was shaken t3iochim. Biophys. ,4cla, 259 (1972) 285 296
287
MOLECULAR WEIGHT OF CHLOROPLAST D N A
at 5-rain intervals over a 3o-min period. The mixture was centrifuged at xo ooo ×g for IO rain at 4 °, and the aqueous layer was removed and dialyzed overnight as above and used for buoyant density analysis in CsC1.
(b) Chloroplast DNA /or electron microscopic examination and sucrose gradient analysis. A suspension of 2 ml of E. gracilis chloroplasts was thawed at 4 °. Sodimn dodecyl sulfate, pronase pretreated by the method of YOUNG AND SINSHEIMER s, and sodium deoxycholate were added to final concentrations of 0.5 o,,jo, I mg/ml, and 1 % , respectively, and the mixture was gently rocked for 5-rain periods at approximately I-h intervals for 12 h at 4 °. NaC10 4 (5 M) was added to I 3I final concentration, and the solution was rocked for 3o rain at 4 ° with an equal volume of buffer-saturated (o.15 M NaCl, o.io M EDTA, o.o5 M Tris-HC1, p H 9.o), distilled phenol and o. 5 volume of chloroform-isoamyl alcohol (24 : I, v/v). The mixture was centrifuged at IOOO × g for 15 min at 4 °. The aqueous phase was removed with a 2-ram bore Pasteur pipette and dialyzed overnight against o.15 M NaC1, O.Ol5 M sodium citrate, o. 5 mM EDTA, p H 8.o, in the cold and then prepared directly for electron microscopy or sucrose gradient analysis. (c) Bacteriophage DNA. 3H-labeled, T 7 DNA was isolated from T 7 bacteriophage grown in the presence of i3H]thymidine 9. Isolated all-labeled T 7 DNA was found to consist of a mixture of whole and half molecules by sedimentation analysis as described by STUDIER 10 a n d BRUNER AND VINOGRAD ll. DNA was isolated from 2 sus O29Cls57 bacteriophage by the methods described by THOMAS AND ABELSON9. DNA was then circularized by the procedure of MACI-IATTIE AND THOMAS ~2.
(d) Other DNA preparations. Escherichia coli bromouracil-labeled hybrid DNA, Microeoccus lysodeikticus DNA, and E. gracilis total cellular DNA were prepared as described previously 5. Electron microscopy Approximately IO/,1 of isolated DNA was added to o.I ml of I M ammonium acetate containing 0.05 °/o cytochrome c and 0.5 % formaldehyde. This solution was spread onto a hypophase of 0. 3 M ammonium acetate containing 0. 5 % formaldehyde or of water with 0.5 % formaldehyde. Preparation of grids, electron microscopy, and contour-length measurements were performed as described previously m3.
Sucrose gradient sedimentation A o.146 to o.585 M linear sucrose gradient (11. 5 ml) containing o.I M NaC1, I mM EDTA, and 0.05 M potassium phosphate, p H 6. 7, was layered into a polyallomer tube at 4 °. A DNA sample containing 0.50 ml of 32p-labeled chloroplast DNA and o.o15 ml of nil-labeled T 7 DNA was layered on top of the gradient and sedimented for 2.5 h at 4 ° ooo rev./min in a Spinco SW 41 rotor at 4 °. Fractions containing 6 drops each were collected from the bottom of the tube through an i8-gauge needle by allowing drops to run gently down the side of the collecting tubes. Gradients were analyzed by electron microscopy and for radioactivity.
CsCl equilibrium density gradient centri/ugation A solution (0.02 M Tris-HC1, p H 8.0) of 3.0 ml was prepared containing Biochim. Biophys. Ac/a, 259 (I972) 285-296
2~8
J.E.
MANNING, O. C. RICHARDS
E. gracilis a2p-labeled chloroplast DNA and E. coli bromouracil-labeled hybrid DNA (O = z.754 g/cm a) and non-radioactive chloroplast I)NA (p = 1.685 g/cm :~) as density markers. CsC1 (3.85 g) was added, and the tube was filled with mineral oil and centrifuged in the Spinco 4o.2 fixed-angle rotor at 35 ooo rev./min for 3 days at 2 5 . Fractions of 4 drops each were collected and diluted with o. 5 ml of o.I 5 M NaCI, o.o15 M sodium citrate, and their absorbance at 2(5o nm and radioactivity were determined. Analytical CsC1 equilibrium density gradient centrifugation was performed as described previously a. Scintillation counting Gradient fractions were prepared for radioactivity measurements bv two methods: (a) Fractions were diluted with o. 5 nfl of o.I 5 M NaC1, o.oI 5 M sodium citrate, 0.05 ml of sahnon sperm DNA (zoo #g), and 0.055 ml of 5.0 M KOH and were incubated at 37 ° for I6 h and then cooled at 4 ~. Cold o.61 M trichloroacetic acid (L43 ml) was added to a final concentration of 0. 3 M. The nfixture was kept on ice for 45 to 6o rain, 2 ml of cold water was added, and the precipitate was collected by vacumn filtration on 24-nun nitrocellulose filters, Type 136 (Selfleicher and Schuell). Filters were washed with two 3-nil portions of cold water, transferred to counting vials, and dried at 80'. For radioisotope determination, Io ml of toluene liquifluor (4.og of 2,5-bis-2-(5-tert.-tmtyl-benzoxazolyl)thiophene to 1 1 of toluene) was added to each vial. (b) Gradient fractions were treated with deoxyribonuclease 1 and snake venom phosphodiesterase by the method of I~.ELI.YANI) SM1TH14. O.50 ml of o.15 M NaC1, O.Ol5 M sodium citrate, 0.o5 ml of sahnon sperm DNA (zoo#g), and o.55 ml of o.6z M trichloroacetic acid were added, and the precipitate was collected and counted as in Method (a).
Reagents Beef pancreatic ribonuclease A, pancreatic deoxyribonuelease I, ribonuclease "1"1, and snake venom phosphodiesterase were obtained from Worthington Biochemical Corp. Salmon sperm DNA and pronase (B grade) were obtained from Calbiochem. Dried M. lysodeikticus cells were purchased from Sigma Chemical Co. Optical grade CsC1 was purchased from Harshaw Chemical Co. Carrier-free [a2P]phosphoric acid was purchased from New England Nuclear Corp.
RESULTS
Examination o] isolated chloroplast DNA in the electron microscope The linear density and molecular weight of circular chloroplast DNA molecules were accurately determined by use of circular bacteriophage 2 DNA as an internal control, a2P-labeled DNA was isolated from chloroplast fractions by gentle lysis with sodium dodecyl sulfate, sodium deoxyeholate, and pronase, followed by deproteinization with a chloroform-phenol mixture. This DNA was added to a solution containing circular ). DNA inolecules, and the mixture was prepared for examination in the electron microscope. Thus, both DNA's were prepared for microscopy under identical conditions. An average contour length of 44.5 (±0.6) #m was obtained for the circular chloroplast DNA and an average contour length of 14. 9 Biochim. Biophys..4cta, 259 (I972) 285 290
DNA
MOLECULAR WEIGHT OF CHLOROPLAST
289
(:ko.3) # m was found for the circular 2 DNA molecules, using 0.3 M ammonium acetate containing 0.5 % formaldehyde as the hypophase in the protein monolayer technique 15 (Fig. I). A picture of a circular chloroplast DNA molecule is shown in Fig. 2. Additionally, rare highly twisted circular molecules were observed (Fig. 3). The contour lengths of the twisted circles, although difficult to determine accurately, were not dissimilar from those obtained for the open circular molecules.
22 12
0
1
o 6 z 4
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,
,
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i
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i
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Fig. ~. F r e q u e n c y distributions of the c o n t o u r lengths of circular D*~A molecules from isolated chloroplast DN"A and from 2 DNA. A m i x t u r e of chloroplast D N A and ). D N A was p r e p a r e d directly for electron microscopy, using a h y p o p h a s e of o. 3 }I a m m o n i u m acetate, o.5 °o forinaldehyde. The circular chloroplast D N A had an average c o n t o u r length of 44.5(±o.6)1,m and is s h o w n hy the cross-hatched area. Circular ~, D N A had a c o n t o u r length of I 4 . 9 ( - - o . 3 ) / m L illustrated by the area b o u n d e d b y h e a v y lines.
32P-labeled DNA from the same preparation of chloroplast DNA was spread with circular ), DNA onto a hypophase of water containing o. 5 °o formaldehyde. The DNA's were picked up on carbon-coated formvar grids and examined in the electron microscope. An average contour length of 48.9 ( ~ i . 2 ) / , m was obtained for the circular chloroplast DNA and an average contour length of 16.2 ( ~ o . 4 ) # m ,,,as found for the circular 2 DNA molecules. Thus, identical ratios of 3.0 for the the contour lengths of circular chloroplast DNA to circular 2 DNA were obtained for both conditions of spreading DNA. Comparison of the length measurements of the two circular DNA's allowed the best calculation of the molecular weight of the chloroplast DNA. The upper densitometer tracing in Fig. 4 shows the distribution of DNA extracted from whole cells of E. gracilis, strain Z, and shows minor peaks with buoyant densities of 1.69o and 1.685 g/cm ~, representative of mitochondrial and chloroplast DNA, respectively6'~6'tL DNA isolated from the same 3~P-labeled chloroplast fractions used for electron microscopy was banded in a CsC1 equilibrium density gradient. This DNA formed a band with a main peak at 1.685 g/cm 3 (Fig. 4, lower tracing), showing that the predominant DNA species is chloroplast DNA. A minor component Biochi~n. Biophys..dora, 259 (I972) 2 8 5 296
290
J. F.. MANNING, O. C. RICHARDS
Fig. 2. Electron micrograph of a rotary shadowed circular I_)•A molecule isolated from a chloroplast fraction from cells of Et~gh'na gracilis, strain Z. The molecule is 44 ,ran ill contour length; the line represents I/tin.
is o b s e r v e d w i t h a b u o y a n t d e n s i t y of 1.698 g / c m 3 ; t h i s D N A h a s b e e n s h o w n t o b e a p o r t i o n of c h l o r o p l a s t D N A e n r i c h e d in c i s t r o n s f o r c h l o r o p l a s t r i b o s o m a l R N A 18. This DNA has been observed previously, but the small quantity makes it unlikely that this DNA accounts for the circular molecules observed in our chloroplast pre-
tliochiln. 14iophyx, ..tcta, 259 (It~72) 285 296
MOLECULAR WEIGHT OF CHLOROPLAST
DNA
29I
Fig. 3. Electron m i c r o g r a p h of a r o t a r y shadowed, t i g h t l y twisted circular molecule of D N A isolated from a chloroplast fraction from cells of E. gracilis, strain Z. The line represents i ,urn.
Biochi~. Biophys..4cta, 259 (1972) 285 296
292
J.v.
MANNING, O. C. I(ICHARDS
E
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i
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8u0y0ni density (g/cm5} Fig. 4- ] ) e n s i t o l n e t e r tracings of p h o t o g r a p h s after equilibriuln d e n s i t y g r a d i e n t c e n t r i f u g a t i o n i11 CsC1 of whole cell D N A (44 Hg, top tracing) a n d of isolated chloroplast D N A (-~ iig, b o t t o m tracinR). M. kvsodeikticus D N A (p 1.731 g/ell] 3) served as t h e reference I)NA.
p a r a t i o n s L l ; u r t h e r m o r e , MANNING t't al. 1 have shown t h a t tile circular molecules are not due to c o n t a m i n a t i o n b y l n i t o c h o n d r i a l D N A or b y other organisms. Thus, the circular D N A molecules are a t t r i b u t a b l e to chloroplast D N A .
Sucrose gradient sedimentation S e d i m e n t a t i o n of carefully isolated 32P-labeled D N A from chloroplasts t h r o u g h sucrose d e n s i t y g r a d i e n t s was used to s e p a r a t e classes of D N A molecules of different m o l e c u l a r weights. The s e d i m e n t a t i o n rate allowed a m e a s u r e m e n t of the size of chloroplast D N A i n d e p e n d e n t of t h a t o b t a i n e d by' electron microscopy. Molecular wholes a n d halves of a l l - l a b e l e d T 7 D N A were used as i n t e r n a l molecular weight m a r k e r s a n d allowed a c c u r a t e measure of the m o l e c u l a r weight of chloroplast D N A . A f t e r s e d i m e n t a t i o n t h e r a d i o a c t i v i t y of the different fractions was d e t e r m i n e d . Three distinct, r a p i d l y s e d i m e n t i n g a2p p e a k s were o b s e r v e d (Fig. 5); a s c a t t e r i n g of a'~p was found at the t o p of the t u b e a n d is not pictured. The relationship of distance t r a v e l e d in the g r a d i e n t to molecular weight, d e v e l o p e d b y BUR(;I AND HERSHEY 19 a n d modified b v I:REH:EI~II~:R~°, was used to d e t e r m i n e the molecular weights of m a t e r i a l in p e a k regions relative to T 7 D N A . Using a value of 2.52 • I o v for the molecular weight of the 3H-labeled T 7 D N A , a m o l e c u l a r weight of 9.2 • I o v was c a l c u l a t e d for the fastest sediinenting 3~p peak ( F r a c t i o n 3 i ) . Material in t h e second fastest sedim e n t i n g 32p p e a k (Fraction 40) corresponds to molecules h a v i n g an average molecular weight of 5.3 " IoL D N A i s o l a t e d from these '~"P-labeled chloroplast fractions was centrifuged in a p r e p a r a t i v e CsC1 equilibrium d e n s i t y g r a d i e n t with n o n - r a d i o a c t i v e chloroplast D N A a n d E. coli b r o m o u r a c i l - l a b e l e d h y b r i d D N A as b u o y a n t d e n s i t y m a r k e r s (l:ig. 0). The p e a k of :~zP-labeled D N A coincides with n o n - r a d i o a c t i v e m a r k e r chh)rophlst D N A from E. gracilis showing t h a t the a2p counts o b s e r v e d in tile sucrose g r a d i e n t ]¢iockim. H i o p k y s . . 4 c t a , -'50 (~(17") : , S 5 : 0 0
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Fraction No.
Fig. 6. Equilibrium d e n s i t y profile of a2P-labeled chloroplast D N A in CsCI d e n s i t y gradient. a2p-labeled D N A was isolated from purified chloroplasts of E. gracilis, strain Z, and nlixed with E. coil bromouracil-labeled h y b r i d D N A (p = 1.754 g/cnl s, n o n - r a d i o a c t i v e peak in F r a c t i o n s 18 to 25) and with chloroplast D N A (p -- 1.685 g/cm a, non-radioactive peak in Fractions 64 to 7 o) as d e n s i t y references. Centrifugation was performed in a neutral CsC1 density gradient at 35 ooo rev./min at 257 for 65 h in a Spinco 4o.2 fixed-angle rotor. Drop fractions were collected and their a b s o r b a n c e and r a d i o a c t i v i t y were determined. @ - - - O , 32p counts/min; 0 - 0 , absorbance at 260 nm, reference DNA; A - A , b o y a n t density. (Fig. 5) a r e p r e d o m i n a n t l y i n c h l o r o p l a s t D N A . F u r t h e r m o r e , t h e a c i d - p r e c i p i t a b l e , a l k a l i - s t a b l e r a d i o a c t i v i t y i n F r a c t i o n s 28 t h r o u g h 6 o o f t h e s u c r o s e g r a d i e n t ( F i g 5) can be completely removed by treatment with deoxyribonuclease and snake venom phosphodiesterase. Riochim. P, iophys..4cta, z59 (i972) 285 296
294
J.E.
MANNING, O. C. RICHARDS
Electron microscopy o~ peak/factions/rom the s~crose gradient Sedimentation of 32P-labeled chloroplast D N A was performed in a sucrose gradient using conditions identical to those of Fig. 5. Every third fraction was analyzed for radioactivity and a pattern similar to Fig. 5 was obtained. The two fractions on either side of the peak fractions were diluted with o.5o ml of o.I 5 M NaC1, o.oz5 M sodium citrate and combined. Preparations of DNA from the fastest sedimenting peak (Fig. 5, Vracti~ns 3o and 32) contained 3 ° ~i~ circular molecules which had an average contour length of 43.5 ( q ~.2)/~m (Fig. 7, area under dashed lines). I.inear molecules varied between 2 and 44 ktm (cross-hatched area) with 74 'I;, of all D N A molecules varying between 4 o and 45/~m. i8 0o of the DNA had contour lengths between z 4 and 27/tin with an average contour length of iq. 7/~m. 16
l
i
,
,
,
,
i
,
i
,
,
,
l
,
,
,
i
i
i
i
I
10
4
2
10
20
Length(~m)
30
40
Fi~. 7. F r e q u e n c y d i s t r i b u t i o n s of c i r c u l a r a n d l i n e a r I ) N , \ mol e c ul e s from t he f a s t e s t a n d second f a s t e s t s e d i m e n t i n g 321) p e a k s in t h e sucrose g r a d i e n t (Fig. 5), The l e n g t h d i s t r i b u t i o n ol l i n e a r D N A m o l e c u l e s fotmd in the f a s t e s t s e d i m e n t i n g ~2p p e a k is s h o w n b y t h e c r o s s - h a t c h e d area, a n d t h e l e n g t h d i s t r i b u t i o n for c i r c u l a r D N A m o l e c u l e s found in t h e s a m e p e a k is s h o w n b y t h e a r e a u n d e r t h e d a s h e d line. The a r e a b o u n d e d b y h e a v y lines s how s t h e l e n g t h d i s t r i b u t i o n of t h e l i n e a r D N A mo lecules found in the second f a s t e s t s e d i m e n t i n g 32p p e a k ; no c i r c u l a r D N A m olecules were o b s e r v e d in t h i s DNA. i " r e q u e n c y d i s t r i b u t i o n s are e x p r e s s e d as t h e p e r c e n t a g e of t h e t o t a l l e n g t h of ] ) N A e x a m i n e d in each peak. The t o t a l l e n g t h of D N A m e a s u r e d from t h e f a s t e s t s e d i m e n t i n g D N A w a s i 3 I 3 p m of w h i c h 3 ° °/o w a s in t h e form of circles. The t o t a l l e n g t h of I ) N A i n e a s u r e d from t h e second fa,stest s e d i m e n t i n g D N A w a s 95r/,,m.
Electron microscopic examination of the D N A molecules in the second fastest sedimenting peak (Fig. 5, Fractions 39 and 41) showed linear molecules having lengths between I and 3o/~m (Fig. 7, area bounded b y h e a v y lines) with 76.5 o~ of the DNA ranging in length from 14 to 27/ml. The D N A in this peak had an average contour length of 19. 7 #m. No circular molecules were observed.
l~i,;chim. Hiophys. _qcta, 250 (3972 ) 2~ 5 zq6
MOLECULAR WEIGHT OF CHLOROPLAST
DNA
295
DISCUSSION
The results indicate that at least one-third of the DNA from chloroplasts of
Euglena gracilis is in the form of circular molecules with an average contour length of 44.5 (~o.6)/~m under our conditions of electron microscopy. Covalently closed DNA molecules in the configuration of a supercoil were also observed; this is the first demonstration of DNA supercoils in chloroplast preparations. Since no linear molecules longer than the longest circle were observed, the DNA molecules in chloroplasts of E. gracilis in silu are (a) circular molecules of about 44/zm, or (b) hoth circular molecules of approximately 44/~m and linear molecules. At the present time it is not possible to favor one of the above two conclusions. The contour length of circular chloroplast DNA was accurately calibrated using circular 2 DNA for an internal standard on the same grids for the electron microscope. The average contour lengths for the circular 2 and circular chloroplast DNA were I4.9 # m and 44.5/,m, respectively, when the hypophase was o. 3 M amnmnium acetate containing o.5 °'o formaldehyde. Using 3.o6. IO7 for the molecular weight of 2 DNA 2°, chloroplast DNA has a molecular weight of 9.2 • IoL A mass per unit length of 2.o6 • IOn daltons per # m is obtained and compares well with that of 2.o 7 • IO6 daltons per/zm obtained by LANG~for duplex DNA examined under similar conditions. Stretching or contraction of DNA molecules caused by variation of the hypophase onto which the molecules are spread appears to be proportional to the length of the molecules. For example, whether the hypophase was water and o.5 °5 formaldehyde or o.3 M ammonium acetate and o.5 °'o formaldehyde, the same ratios of contour length of circular chloroplast DNA to circular 2 DNA were obtained. Thus, use of an internal standard in determination of the mass per unit length of DNA molecules is both essential and meaningful. Analysis of carefully isolated chloroplast DNA by sucrose gradient sedimentation showed that the DNA distinctly separated into three rapidly sedimenting fractions. The fastest fraction had an average molecular weight of 9.2 • lO7, as calculated from the sedimentation coefficient of 50 S 2°. This value for the molecular weight obtained by sedimentation techniques is the same as the molecular weight calculated from the average contour length of the molecules observed in the electron microscope. No increased frequency of circular molecules was observed in this fastest sedimenting peak; this m a y be a result of the increased manipulations involved in analyzing the sucrose gradient. Furthermore, no separation of circular and linear molecules of Minilar contour lengths was observed in the sucrose gradient, The average molecular weight of molecules in the second fastest peak was calculated from the sedimentation coefficient and was found to be 5.3 " ioL 76 °,o of the DNA in this peak had contour lengths between 14 and 27/~m, the lengths expected for shear products of whole molecules of chloroplast DNA broken into molecular halves. The molecular weight of chloroplast DNA using internal molecular weight standards for electron microscopy and for sedimentation analysis has been shown to be 9.2 " x& and revises the earlier estimate of 8.3 • xo7 (ref. x) based solely on electron microscopy. The accurate determination of the molecular weight of the chloroplast DNA molecule using two independent methods does, however, confirm our previous interpretation that multiple copies of this DNA molecule exist per chloroplast.
Biochim. Biophys. Xcta, 259 (1972) 285-296
296
j . F . . MANNING, o. C. RICHARDS
NOTE ADDED 1N PROOF: ( R e c e i v e d Cospreading that DNA
of 2 bacteriophage
o n e u n i t l e n g h t of 2 D N A
lenght.
Therefore,
February
less than
DNA
was mixed o.I
'~?, o f t h e
2nd, 1972 ) with chloroplast with
about
DNA
was performed
o n e c i r c l e of 4 4 - # m
circles observed
could
so
contour
be triple-lenght
molecules.
ACKNOWLEDGMENTS
This research was supported by Grant GB-2o763 from the National Science Foundation. One of us (J.E.M.) was supported by U.S. Public Health Service Training Grant GM-ooI52. We should like to thank I)r. D. R. \,Volstenhohne for use of his electron microscope and facilities. REFERENCES i J. E. MANNING, D. 14.. \\'OLSTI'2NHOLME, [{. ,~. RYAN, J, :\. HUNTER ANI) 1). C. RICHARDS, Proc. Natl. d c a d . Sci. U.S., 68 11971) II69. 2 E. STUTZ, FI'21IS I.ett., S (~97 o) 25. 3 J. (k WETMUR AND N, 1)AVIDSO.X, .f. ),lol. Biol., 31 (I06~) 3413. 4 D. LANG, f . Mol. Biol., 54 (~97 °) 557. 5 ()- C. I(ICHARDS, H. S. I(YAN AND J. E. ~[ANNIN(;, t3i,chim, l~iophys..qcta, 23 s (I97~) ~0o. 0 G. ~3RAWF.RMXN AND J. M. EIS~NSTAI)T, Hiochim. l¢i@hys, dcta. 0~ 119643 4777 J. MARMt:R, ,[. i~Icd. 13iol., 3 (1901 ) 2o8. S E . T. YOUNG AND 1{. 1.. SINSHEIIMGR, .l- d~'/0/, l]iol., 3 o (10(37) l05. 9 C. A. THOMAS AND J. ABELSON, ill (;. l.. CANTONI ANI) ]). 1(. I)AVIES, Procedures i~ Nucleic .4cid lfcsearck, H a r p e r a n d Row, Nexx York, 1960, p, 553io 1:. \V. STUDIO:R, .[. Mol. I~iol., 11 11005) 373. t l R . I~RUNER AND J. \rlNOGRAI), lliochim. Biophys..qcta, toS (1065) i8. 12 1.. A. MAcH.¢TTIE AND C. A. "I'Ho.~IAS, Scie~wC, i44 [t004) ,142. 13 1). 1<. \¥OLST~NltOL.XI~; AND N. J. (;ROSS, Proc. N a t l . . 4 c a d . Sci. U..q., ol (,gO,S) 245. 14 T . J. KELLY AND H. (). SMIT,L J. Mol. Biol., 5i (I97 °) 393. 15 ;\. K. KLI~:INSCHMIDT aND R. K. ZAHN, Z. Natur/orsch., 14b (1059) 77 ° • 10 D . S. RAY AXD 1'. C. HANAWALT, .1. 310l. 13iol., 1~ (I965) 76(3. 17 M. EDEL.XL.XN, J. A. St'HIFF AND H. T. EPSTI~IN, ,J. ]fol. Biol., i~ (,0o5) 700 . 18 E. STUTZ ANI) J. P. VA~DREY, F.I'2tLq Lell., 17 ([97 ~) 277. t0 E. BURGI AND ;\. D. [{F,RSIII*:}', t3iopkys. ,[., 3 ([903) 3o0 • 2o D. I:REIIq~:LDt,'R, .1. 3Ioi. l]iol., 54 (I97 o) 507 . 13iochim. ]~iopk3's..qcla, 259 (I072) 2~ 5 20()