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Hydroxyapatite chromatography of deoxyribonucleic acids from Euglena gracilis
BIOCHIMIE, 1972, 54, 1013-102I.
Hydroxyapatite chromatography of deoxyribonucleic acids from Euglena gracilis. E. STUTZ and G. BERNARDI.
Laboratoire de G~t~tique Moldct:laire, Institut de Bioloyie Moldculaire, Paris 5", Fral~ce and Department o[ Bioloyical Sciences, Northweslern University, Evanston, Ill. 60 201. (3/6/1972). Summary. -- The chromatographic behavior of native Euglena 9racilis DNA's on hydroxya,patite columns has been investigated. Total nucleic acid preparations can be f'ractionated by column chromatography to yield nuclear DNA and organeHe (mitoehondrial + chloroplast) DNA. A batch procedure permits the rapid separation of organeHe DNA from ,total nucleic acid preparations. Chromatography of DNA preparations from heat-bleached mutants permits the separation of mitochondrial DNA from nuclear DNA. Chromatography of DNA preparations from chloroplast-enriched fractions of Euglena permits the separation of ch,loroplast DNA, and in addition, of a satellite DNA previously described (~ = 1.700 g/emS). Some physical and chemical properties of ~he Euglena DNA's, as obtained by chromatography on hydroxyapatite, are reported.
INTRODUCTION. T h o u g h mostly used for the s e p a r a t i o n of native a n d d e n a t u r e d DNA [1, 2], h y d r o x y a p a t i t e (HA) c o l u m n s have a c o n s i d e r a b l e d i s c r i m i n a t o r y p o w e r t o w a r d s different native DNA structures [3, 4q. Not t a k i n g into c o n s i d e r a t i o n the case i n w h i c h the s e c o n d a r y (single-stranded DNA from ¢ X 174 phage) or the t e r t i a r y structure (twisted c i r c u l a r DNA from p o l y o m a virus) are grossly different and cause a different c h r o m a t o g r a p h i c behavior [3, 4], it is b e c o m i n g i n c r e a s i n g l y evident that differences i n nucleotide sequences i n otherwise s i m i l a r DNA's (the s i m i l a r i t y c o n c e r n ing the double-strandedness, the m o l e c u l a r weight, the linear, or open circular, configuration, and also the n u c l e o t i d e composition) m a y be sufficient to d e t e r m i n e different elution molarities. Recent w o r k [5-71 suggests that DNA's c o n t a i n i n g short repetitive sequences like satellite DNA's may, in general, show p a r t i c u l a r elution molarities. A p a r t i c u l a r l y s t r i k i n g case is that of m i t o c h o n drial DNA's from wild-type a n d cytoplasmic (> m u t a n t yeast (Saccharomyces cerevisiae) ceils [8-10]. These DNA's, w h i c h are characterized by very low GC contents ( r a n g i n g from 18 p. cent in w i l d - t y p e cells to only 4 p. cent i n some cytoplasmic <> mutants), are elnled at higher phosphate molarities c o m p a r e d to yeast n u c l e a r I)NA's or bacterial DNA's. The higher
elution molarities of yeast m i t o c h o n d r i a l DNA's are due to the presence i n these DNA's of long AT-rich stretches [10] c o n t a i n i n g both a l t e r n a t i n g dAT : dAT and n o n - a l t e r n a t i n g dA : dT s t r u c t u r e s [11]. I n fact, poly ( d A T : dAT) a n d poly ( d A : dT) a n d the dAT-rich satellite DNA from Cancer pagurus, elute at slightly a n d m u c h higher molatitles, respectively, than bacterial DNA's [10] ; finally e x p e r i m e n t s on e n z y m a t i c a l l y degraded yeast m i t o c h o n d r i a l DNA's have s h o w n that GCrich fragments elute at a l o w e r phosphate molarity compared to the AT-rich fragments [12, 13]. The results with yeast m i t o c h o n d r i a l DNA p r o m p t e d a s i m i l a r k i n d of e x p e r i m e n t s with Euglena qracilis chloroplast a n d m i t o c h o n d r i a l DNA w h i c h both are low in their GC contents (24 p. cent a n d 32 p. cent, respectively) [14]. These DNA's might share some s t r u c t u r a l features w i t h the m i t o c h o n d r i a l DNA's from yeast a n d display, therefore, a s i m i l a r c h r o m a t o g r a p h i c behavior. F r o m a p r a c t i c a l p o i n t of view, tile s e p a r a t i o n of organellar from n u c l e a r DNA i n the case of Euglen.a is complicated by the fact that the former only represents 1-2 p. cent of the total cellular DNA, a level about 10 times lower t h a n that of yeast n l i t o c b o n d r i a l DNA. If successful, however, the method w o u l d lend itself to scale tip the prep a r a t i o n of o r g a n e l l a r DNA in a relatively simple a n d i n e x p e n s i v e way, a p o i n t of c o n s i d e r a b l e im-
E. Stutz and G. Bernardi.
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p o r t a n e e in v i e w of the c u r r e n t i n t e r e s t in the b i o l o g i c a l f u n c t i o n of b o t h DNA's [151. In the f o l l o w i n g w e r e p o r t the c h r o m a t o g r a p h i c s e p a r a t i o n of b o t h Euglena c h l o r o p l a s t a n d mitoc h o n d r i a l DNA u s i n g e i t h e r total c e l l u l a r n u c l e i c a c i d s or o r g a n e l l a r DNA's e n r i c h e d s a m p l e s as s t a r t i n g materials. In a d d i t i o n , the p r o c e d u r e used p e r m i t s the s e p a r a t i o n of n u c l e a r DNA and of a satellite DNA. This w o r k has been p r e s e n t e d at the Colloque de la Soci6t6 de C h i m i e Biologique sur les Acides D ~ s o x y r i b o n u c l ~ i q u e s des E u c a r y o t e s (Strasbourg, March 16-18, 1972). MATEI~IALS AND METHODS. 1) Cultivation of cells. Euglena gracilis, Klebs (Z-strain ; Culture Coll e c t i o n of Algae, I n d i a n a U n i v e r s i t y , N ° 753) w a s g r o w n a u t o t r o p h i c a l l y , h a r v e s t e d a n d s t o r e d as r e p o r t e d e a r l i e r [16]. Euglena gracilis, heat-bleac h e d mutant, w a s g r o w n h e t e r o t r o p h i c a l l y on Euglena B r o t h (Difco) either in light o r in d a r k . The m u t a n t was i s o l a t e d in E v a n s t o n a n d g r o w n for m o r e t h a n 12 m o n t h s w i t h o u t s h o w i n g a n y green i n g c a p a c i t y . No c h l o r o p l a s t DNA (0 = 1.685 g/ c m 3) could be d e t e c t e d in this mutant. 2) Parification of chloroplast and mitochondria. C h l o r o p l a s t s w e r e i s o l a t e d f r o m w i l d - t y p e cells as r e p o r t e d [16], a n d p u r i f i e d b y flotation in a L u d o x g r a d i e n t [17]. M i t o c h o n d r i a w e r e i s o l a t e d f r o m b l e a c h e d cells w h i c h w e r e b r o k e n u s i n g a F r e n c h P r e s s at 2 000 psi, in 0.25 M sucrose, 0.01 M tris HC1, p H 7.9, 0.25 M KC1, 0.00,4 M MgCl 2 a n d 0.005 M ~ - m e r c a p t o e t h a n o l . The c r u d e mitoc h o n d r i a w e r e o b t a i n e d b y d i f f e r e n t i a l centrifugation a c c o r d i n g to K r a w i e c a n d E i s e n s t a d t [181. 3) Nucleic acid extraction. Total n u c l e i c acid. 60 g (wet p a c k e d ) w i l d t y p e cells w e r e r e s u s p e n d e d in 0.15 M NaC1 cont a i n i n g 2.5 p. cent s o d i u m d o d e c y l s u l f a t e (SDS) (150 ml). The m i x t u r e w a s a d j u s t e d to 1 M NaCl w i t h NaC1 c r y s t a l s and s t i r r e d o v e r n i g h t at 4°C. The s u s p e n s i o n w a s c e n t r i f u g e d for 20 rain at 27,000 × g (Sorvall SS-34) a n d the s u p e r n a t a n t p r e c i p i t a t e d w i t h 1 volume of c o l d ethanol. The p r e c i p i t a t e was r e c o v e r e d b y s p i n n i n g at 27,000 × g, for 20~ m i n (Sorvall SS-34), r e d i s s o l v e d in 0.01 M NaC1 (50 ml), a d j u s t e d to 1.5 p. cent SDS, 0.15 M NaC1 a n d s t i r r e d for 30 rain. After adiust e m e n t of NaC1 to 1 M and r e p e t i t i o n of s t i r r i n g o v e r n i g h t at 4°C, the s u s p e n s i o n w a s again centrifuged at 27,000 × g, for 20 rain a n d the s u p e r n a t a n t p r e c i p i t a t e d w i t h 1 volume of ethanol. The
BIOCHIM1E, 1972, 54, n ° 8.
r e c o v e r e d p r e c i p i t a t e w a s dissolved in 0.15 M NaCI (15 ml) a n d the solution e x t r a c t e d w i t h chlor o f o r m - i s o a m y l alcohol ( 2 4 : 1 ; v/v). The chlorof o r m e x t r a c t i o n was r e p e a t e d 3 times a n d the final aqueous p h a s e w a s d i a l y z e d against 0.01 M s o d i u m p h o s p h a t e buffer a n d used for h y d r o x y a p a t i t e c h r o m a t o g r a p h y (total A260 units : 10'21). In some eases the n u c l e i c a c i d e x t r a c t was t r e a t e d w i t h r i b o n u c l e a s e as specified b e l o w (Section 4). 4) DNA from chloroplasts and mitochondria. The p u r i f i e d organelles from 50 g of e i t h e r green or b l e a c h e d cells w e r e r e s u s p e n d e d in 10 ml of 0.1 M tris-HCl (pH 7.5), 2.5 p. cent SDS, 0.01 M EDTA, a n d h e a t e d for 10 rain to 60 °. The suspension w a s a d j u s t e d to 1 M NaC104 a n d e x t r a c t e d t w i c e w i t h 2 volumes of c h l o r o f o r m - i s o a m y l alcohol ( 2 4 : 1 ; v/v). The final aqueous l a y e r w a s digested w i t h BNAase ( r i b o n u c l e a s e B, bovine p a n creas, t y p e VII, Sigma) r e e x t r a e t e d a n d d i a l y z e d o v e r n i g h t against 0.1 M NaC1-0.05 M s o d i v m phosp h a t e buffer, p H 6.8, 0.1 M EDTA. The d i a l y z a t e was p u r i f i e d on a m e t h y l a t e d s e r m n a l b u m i n k i e s e l g u h r (MAK) [191 column. The DNA p e a k w a s p o o l e d a n d d i a l y z e d against 0.1 × s t a n d a r d saline c i t r a t e (SSC ; 0..15 M NaC1, 0.0,15 M citrate, p H 7.5). 1.2 to t.5 A2s0 units w e r e r e c o v e r e d f r o m green c e l l s ; 10-15 A2so units f r o m b l e a c h e d mutant. The degree of c o n t a m i n a t i o n of c h l o r o p l a s t DNA b y n u c l e a r DNA v a r i e d from p r a c t i c a l l y n o t h i n g to about 60 p. cent. The m i t o c h o n d r i a l D'NA p r e p a r a t i o n used h e r e c o n t a i n e d 90 to 95 p. cent of n u c l e a r DNA. 5) Chromatography on hydroxgapatite. Column a n d b a t c h p r o c e d u r e s w e r e p a t t e r n e d after the e x p e r i m e n t s d e s c r i b e d e l s e w h e r e on y e a s t m i t o c h o n d r i a l DNA [10]. 6) Ultracentrifugation experiments. S e d i m e n t a t i o n v e l o c i t y e x p e r i m e n t s a n d analytical caesium chloride density-gradient experim e n t s on native a n d d e n a t u r e d DNA w e r e done as d e s c r i b e d e l s e w h e r e El0]. P r e p a r a t i v e c a e s i u m c h l o r i d e d e n s i t y g r a d i e n t e x p e r i m e n t s w e r e also p e r f o r m e d as d e s c r i b e d e l s e w h e r e [61. Denaturation- renaturation experiments were p e r f o r m e d as follows : DNA s a m p l e s (2-3 ~Jg in 0.5 ml) in 0.05 M NaC1 w e r e d e n a t u r e d b y h e a t i n g at 100 ° for 10 min, f o l l o w e d b y q u e n c h i n g in an ice bath. D e n a t u r e d s a m p l e s w e r e r e n a t u r e d b y a d j u s t i n g the solvent to 2 × SSC w i t h 10 × SSC a n d b y k e e p i n g t h e m f o r 4 h o u r s at 64 °. 7) Base composition of DNA. This w a s d e t e r m i n e d b y e n z y m i c a l l y d e g r a d i n g DNA to n u c l e o s i d e s and b y a n a l y z i n g the l a t t e r
Hydroxyapatite chromatography of E u g l e n a o n e i t h e r B i o Gel P-2 c o l u m n s a c c o r d i n g to P i p e r n o a n d B e r n a r d i [20] o r A m i n e x A6 c o l u m n s as d e s c r i b e d b y T h i e r y , E h r l i c h a n d B e r n a r d i [21].
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bed, except that no RNA peak was evident ; some low-eluting material, probably formed by residual, d e g r a d e d RNA, w a s f o u n d i n t h e s e p r e p a r a t i o n s .
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FIG. 1. - - C h r o m a t o g r a p h y of a t o t a l nucleic acid extract of Euglena gracilis. 150 ml of nucleic acid solution in 0.01 M sodium p h o s p h a t e buffer (150 A~0 units) were loaded on a 1.08 × 60 cm h y d r o x y a p a t i t e column e q u i l i b r a t e d w i t h t h e same buffer. 1'00 ml of this buffer were used to w a s h the column and a 0.01 to 0.5 M g r a d i e n t of sodium p h o s p h a t e buffer, pH 6.8 (1200 ml) w a s applied. 3.4 ml fraction were collected. Flow rate was 34 re,l/hr. Circles indicate 'the l i m i t s of the f r a c t i o n s w h i c h were pooled together.
200
FRACTION NUMBER
RESULTS. Figure 1 shows the hydroxyapatite chromatog r a m of a t o t a l n u c l e i c a c i d e x t r a c t f r o m Eufflena yracilis. T h e f i r s t b i p h a s i e p e a k is f o r m e d b y R N A w h i c h is w e l l s e p a r a t e d f r o m t h e s e c o n d m a j o r p e a k r e p r e s e n t i n g DNA. 2,1.7 A~60 u n i t s w e r e r e c o v e r e d u n d e r t h e D N A p e a k , w h i c h is a p p r o x i m a t e l y 15 p. c e n t of t h e t o t a l A~60 i n p u t . T h i s r o u g h l y c o r r e s p o n d s to t h e D NA : R N A r a t i o f o u n d i n Euglena w i l d - t y p e c e l l s [23J. T h e b u l k of t h e D N A e l u t e d at a p h o s p h a t e c o n c e n t r a t i o n of 0.28 M. T h e fractions under the DNA peak were pooled in four D N A s a m p l e s a, b, c, d, w h i c b , a f t e r c o n c e n t r a t i o n and dialysis, were analyzed for their buoyant dens i t i e s i n n e u t r a l CsCA. T h e f o u r p r o f i l e s a r e s h o w n in figure 2 and though the gradients were overl o a d e d i n c a s e a, b, a n d c, n o s a t e l l i t e D N A w a s detectable. It was only in sample d that a compon e n t of l o w e r b u o y a n t d e n s i t y (~ = 1.6,85 g/cm,~) s h o w e d u p i n a d d i t i o n to t h e m a i n n u c l e a r D N A (~ ~ 1.707 g / c m 3 ) . T h e 1.685 g / c m a b u o y a n t d e n s i t y is t y p i c a l f o r Euglena c h l o r o p l a s t D N A [23, 24, 25]. T h e m i t o c h o n d r i a l D N A w i t h a r e p o r t e d d e n s i t y of 1.690 g / c m a [26, 27] c o u l d n o t b e disc e r n e d i n a n y of t h e s e p r o f i l e s . T h e s a t e l l i t e p e a k r e p r e s e n t e d 15 p. c e n t of t h e n u c l e a r p e a k i n p r o file d a n d s o m e w h a t l e s s t h a n 1 p. c e n t of t h e t o t a l DNA. T h e c h r o m a t o g r a p h i c b e h a v i o r of t o t a l n u c l e i c acid preparations which had undergone RNase t r e a t m e n t w a s e x a c t l y t h e s a m e as t h a t just d e s c r i -
BIOCHIMIE, 19:72, 54, n o 8.
T h e f a c t t h a t t h e o r g a n e l l e D N A is e l u t e d at t h e highest molarity in the chromatogram is v e r y
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1.685 I 1.707 I t.742 FiG. 2. - - Seannings of pooled DNA fractions f r o m the h y d r o x y a p a t i t e c h r o m a t o g r a m of figure 1. a) 147-156 (280 nm) ; b) 157-181 (28[} n m ) ; c) 182190 (280 nm) ; d) 191-220. Samples were centrifuged at 44,000 r e v / m i n for 20 h r at 25°C in a n e u t r a l Cs'CI density gradient. DNA from phage 2 C (1.742 g/cm~) was used as a density marker. Scannings were done at 265 nm, u n l e s s otherwise indicated by a n u m b e r in brackets a f t e r the fraction number.
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f a v o r a b l e for i s o l a t i n g this c o m p o n e n t u s i n g a b a t c h p r o c e d u r e [10]. K n o w i n g f r o m the first exp e r i m e n t that the bulk of n u c l e a r DNA was eluted w i t h 0.28 M s o d i m n p h o s p h a t e buffer, 50 ml of hyd r o x y a p a t i t e , p r e v i o u s l y e q u i l i b r a t e d w i t h 0.28 M p h o s p h a t e , w e r e s u s p e n d e d in a total v o l u m e of 100 ml of 0.28 M p h o s p h a t e c o n t a i n i n g 871 A ~ 0 units of total n u c l e i c acid. The suspension was gently s t i r r e d o v e r n i g h t at r o o m t e m p e r a t u r e . T h e HA crystals w e r e w a s h e d t h r e e times w i t h 50 mlv o l u m e s of 0.28 M p h o s p h a t e and l a y e r e d on a 2 × 60 cm h y d r o x y a p a t i t e c o l u m n e q u i l i b r a t e d w i t h the same buffer. T h e c o l u m n was w a s h e d w i t h 80 ml of 0.28 M buffer and eluted w i t h a l i n e a r 0.28 to 0..48 M (1,920 nil) m o l a r i t y g r a d i e n t of phosphate. The flow rate was 6.8.5 m l / h . F r a c tions of 5.7 nfl w e r e collected. U n d e r these conditions, 3.25 A.260 units w e r e eluted ( c h r o m a t o g r a m not s h o w n ) . T h r e e DNA samples w e r e o b t a i n e d by p o o l i n g f r a c t i o n s eluted b e t w e e n the f o l l o w i n g p h o s p h a t e m o l a r i t i e s : a, 0.28-0.29 ; b, 0.29-0.30 ; c, 0.30-0.32. T h e r e c o v e r y in a, b, c w e r e 1.0,7, 0.62, 1.56, A260 units, r e s p e c t i v e l y . T h e p o o l e d f r a c t i o n s w e r e c o n c e n t r a t e d by r o t a r y e v a p o r a t i o n , d i a l y z e d
5-10 p. cent, and 70-80 p. cent, r e s p e c t i v e l y . The a m o u n t of this l i g h t e r DNA, c o r r e s p o n d i n g to organelle DNA (see below), e x t r a c t e d by the b a t c h m e t h o d is e q u i v a l e n t to the a m o u n t of organelle DNA f o u n d in the c h r o m a t o g r a m of figure 1. This i n d i c a t e s that the b a t c h m e t h o d gives the same y i e l d as c o l u m n c h r o m a t o g r a p h y , and is in essence feasible for large-scale p r o d u c t i o n of o r g a n e l l a r DNA f r o m Euglena. The p u r i f i c a t i o n of c h l o r o p l a s t DNA f r o m Eaglena cells is c o m p l i c a t e d by the p r e s e n c e of mitoc h o n d r i a l DNA, since the f r a c t i o n eluting at high m o l a r i t y m i g h t c o n t a i n the m i t o c h o n d r i a l DNAas
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FIG. 4. Chromatography of DNA from a mitoehondria-enriched fraction from a bleached mutant of Euglena gracilis. 25 inl of DNA solution in 0.1 M sodium phosphate buffer (2,5.2 A~o units) were loaded on a 1.08 × 60, cm hydroxyapatite column equilibrated with the same buffer. The c(~lumn was washed with 30 ml of O]is buffer and a gradient of 0.1 to 0.5 M sodium phosphate (80,0 lnl) was applied. 4.5 ml fractions were collected. Flow rate Was 28 ml/h. A~eorecovery was 92 p. cent. Circles, indicate the limits of the fractions which were pooled together. -
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1.7o7 I ( 1.742 Fro. 3. - - Seannings of pooled DNA fractions from t h e batch experiment (see Tex,t). a) 40-54 ; b) 55.-68 ; c) 69,-92. The mo]ari.ty of the sodium 'phosphate buffer were 0.28 at fraction 40, 0.29 at fraction 55, 0,30 a tfraction 69,. Gentrifugation conditions in neutral CsCil density gradients as given in figure 2.
against 0.1 M NaCJ a n d aliquots w e r e a n a l y z e d by u l t r a c e n t r i f u g a t i o n in CsC1 density gradients (fig. 3). Sample a c o n t a i n e d only n u c l e a r DNA (1.707 g / c m 3) ; sample b and c c o n t a i n e d , in addition, a l i g h t e r DNA (1.68,5 g / c m 3) at levels of about
BIOCHTMIE, 19.72, 54, n ° 8.
well. T h e profile e (,fig. 3) dose not r e v e a l any c o m p o n e n t at 1.6,90 g f c m 3, w h i c h is the r e p o r t e d b u o y a n t density of Euglena m i t o c h o n d r i a l DNA. T h e r e f o r e , e i t h e r the m i t o c h o n d r i a l DNA eluted at a different m o l a r i t y or the two satellites did not b a n d sufficiently apart. To clarify this point, w e c h r o m a t o g r a p h e d DNA extracted from a mitochondria-enriched fraction o b t a i n e d fronl a h e a t - b l e a c h e d mutant. F i g u r e 4 shows the c h r o m a t o g r a m w h i c h was again separated into four fractions, a, b, c a n d d. The d e n s i t y profiles of these f r a c t i o n s are s h o w n in figure 5. Again, it is only the last f r a c t i o n (buffer c o n c e n t r a t i o n 0.3'0-0.35 M) w h i c h contains the satellite, thougb still h e a v i l y c o n t a m i n a t e d w i t h n u c l e a r DNA. The b u o y a n t d e n s i t y in neutral CsC1 of the m i t o c h o n d r i a l DNA was 1.6,88 g/em3, slightly
Hydroxgapatite chromatography of b e l o w t h e r e p o r t e d v a l u e s [26, 27]. I d e n t i c a l d e n s i t y v a l u e s (1.688 g / e r a 3) w e r e o b t a i n e d , h o w e v e r , for mitochondrial DNA isolated from a bleached m u t a n t [18]. T h e p r o x i m i t y of t h e d e n s i t i e s of chloroplast and mitochondrial DNA probably prev e n t e d t h e r e s o l u t i o n of m i t o c h o n d r i a l f r o m c h l o r o p l a s t D N A i n p r o f i l e c (fig. 3). A d e n a t u r a t i o n -
DNA.
Euglena
1017
mately the same buffer molarity, and therefore the s a t e l l i t e D N A f r o m t h e w i l d - t y p e is a m i x t u r e of chloroplast and mitochondrial DNA. O r g a n e l l e
/\
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Fro. 5. - - Seannings of pooled DNA f r a c t i o n s f r o m the h y d r o x y a p a t i t e e h r o m a t o g r a m of figure 4. a) 31-33 ; b) 34-38 ; c) 39-41 ; d) 42-5,0. Centrifugalion conditions in n e u t r a l CsC1 are given in figure 2. r e n a t u r a t i o n e x p e r i m e n t w i t h s a m p l e c (fig. 3) a n d s a m p l e d (fig. 5) c o n f i r m s t h i s a s s u m p t i o n ; t h e respe~,(ive p r o f i l e s a r e d i s p l a y e d i n f i g u r e 6. T h e D N A f r a c t i o n f r o m t h e w i l d - t y p e d e n a t u r e s as o n e b r o a d p e a k ( p r o f i l e c) (~ = 1.70,0 g / c m 3) w i t h a s h o u l d e r at 1.720 g / c m 3. A f t e r r e n a t u r a t i o n , h o w e v e r , a s h a r p m a j o r p e a k (1.6~87 g / c m a) a n d a n e v i d e n t s h o u l d e r (1.696 g / c m 3) a p p e a r s , i n a d d i t i o n to t h e m i n o r n u c l e a r D N A p e a k (1.717 g / c m 3 ) p r o f i l e c'). T h e s a t e l l i t e f r o m t h e b l e a c h e d m u t a n t s h o w s , a f t e r d e n a t u r a t i o n ( p r o f i l e d), a s h o u l d e r of d e n s i t y 9 = 1.700 g / c m ~ a n d a m a j o r n u c l e a r D N A p e a k (9 = 1.72.0 g / c m 3 ) ; a f t e r r e n a t u r a t i o n ( p r o file d ' ) a s h a r p p e a k (9 ---- 1.696 g / c m a ) r e p l a c e s t h e b r o a d s h o u l d e r of p r o f i l e d a n d a m a j o r n u c l e a r D N A p e a k (~ : 1.717 g / c m 3 ) . C o m p a r i n g p r o f i l e c' w i t h d ' o n e r e a d i l y sees t h a t t h e s h o u l d e r in the former matches the renatured satellite (mit o c h o n d r i a l DNA) of t h e b l e a c h e d m u t a n t . T h e c o n c l u s i o n is t h a t b o t h c h l o r o p l a s t a n d m i t o c h o n driaI DNA elute from hydroxyapatitc at approxi-
BIOCHIMIE, 1972,
54, n ° 8.
I
1.700 1.720
J
1.687
1.696 1.7l
1.742
I 1.742
FIG. 6. - - Scannings of h e a t - d e n a t u r e d and r e n a t u r e d DNA from f r a c t i o n (c) of t h e b a t c h e x p e r i m e n t (see figure 3) a n d f r a c t i o n (d) f r o m e h r o m a t o g r a m of t h e bleached m u t a n t DNA (see figure 4). c and c' are the d e n a t u r e d a n d r e n a t u r e d DNA, respective.ly, from. f r a c t i o n (c) ; d a n d d' are the d e n a t u r e d a n d r e n a t u r e d D.NA from fraction (d), respectively. C e n t r i f u g a t i o n conditions in n e u t r a l CsC1 as given in figure 2.
DNA's should be prepared therefore from bleached mutants for mitochondrial DNA and from chlorop l a s t e n r i c h e d f r a c t i o n s f o r c h l o r o p l a s t DNA. In subsequent work, we investigated the chrom a t o g r a p h i c p u r i f i c a t i o n of c h l o r o p l a s t - e n r i c h e d f r a c t i o n s . I n t h e c o u r s e of t h e s e e x p e r i m e n t s s o m e
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Fro. 7. - - C h r o m a t o g r a p h y of DNA from a chlorop l a s t - e n r i c h e d f r a c t i o n of Euglena gracilis. 7 ml of DNA solution in 0.01M sodium p h o s p h a t e buffer (5.7 A_~o units) were loaded on a 1.08 × 60' em h y d r o x y a p a t i t e column e q u i l i b r a t e d w i t h the same buffer. The column was w a s h e d w i t h 30 ml of this buffer and 0.01 to 0.5 M g r a d i e n t (100'0 ml) was applied. 4.0 ml f r a c t i o n s were collected. Flow rate was 28 m l / hr. A~o recovery was 82 p. cent. Circles indicate the l i m i t s of the fractions w h i c h were pooled together.
E. S t u t z a n d G. B e r n a r d i .
1018
n o v e l i n f o r m a t i o n w a s o b t a i n e d on t h e s a t e l l i t e D N A w h i c h w a s r e c e n t l y d e s c r i b e d to o c c u r in c h l o r o p l a s t - e n r i c h e d f r a c t i o n s f r o m Euglena [17]. Two different chloroplast DNA preparations w e r e used, t h e first o n e still h e a v i l y c o n t a m i n a t e d
d
I I I I I I 1 I bl I
b y n u c l e a r DfNA, t h e s e c o n d o n e w i t h no signific a n t a m o u n t of n u c l e a r DNA (p = 1.707 g / c m a ) . Figure 7 shows the chromatogram obtained with t h e first p r e p a r a t i o n ; t h e e l u t i o n p r o f i l e s h o w s t w o d i s t i n c t p e a k s w h i c h c o r r e s p o n d to n u c l e a r D N A an d c h l o r o p l a s t DNA, r e s p e c t i v e l y . I n fact, this profile almost mimics the buoyant density p r o f i l e of t h e s t a r t i n g m a t e r i a l (fig. 8, a), c o n f i r m i n g t h e r e b y t h a t t h e c o l u m n is q u i t e e f f i c i e n t in s e p a r a t i n g t h e t w o t y p e s of D,NA. T h i s is confirm e d b y t h e b u o y a n t d e n s i t y a n a l y s i s of t h e five f r a c t i o n s b, c, d, e, f, w h i c h is in e s s e n c e g a v e a
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1.685! I 1.705 I I.~2 Fro. 8. - - Seanning s of pooled DNA fractions from the chromatogram of figure 7. The scanning of the starting ma'terial is shown in profile (a). b) 100-10.6 ; c) 107-111) ; d) 111-114 ; e) 115-117 ; f) 118-127. Centrifugation conditions in neutral CsC1 density gradients as given in figure 2. 1.685
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FRACTION NUMBER F1G. 10. - - Chromatography of DNA from chloroplast fraction of Euglena gracilis. 4.6 ml of DNA solution in 0.1 M sodium phos,phate buffer (3.6 A~o units) were loaded on a 1:08 X 60 cm hydroxyapatite column equilibra, ted 'with 'the same buffer (3,0 ml) and a 0.1 to 0.5 M gradient (800 ml) was applied. 5.5. ml fractions were co.llected. Flow rate was 33 m l / h r . A~o units recovery was 95 p. cent. Circles indicate the limits of the fractions which were pooled together.
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FIG. 9. Scannings of heat-denatured b, c, d, e and renatured b', e', d', e, pool.ed DNA fractions from the chromatogram of figure 7. CeI~trifugation conditions in neutral CsC1 as given in figure 2. Pooled fractions are indicated as in figure 8.
BIOCHIMIE, 19,72, 54, n ° 8.
s i m i l a r r e s u l t as t h e first c h r o m a t o g r a m w i t h a p r o p o r t i o n a t e up s h i f t of c h l o r o p l a s t DNA. Again, t h e e a r l y e l u t i n g f r a c t i o n (b, c) c o n t a i n o n l y the n u c l e a r DNA, t h e l a t e r f r a c t i o n s (e, f), c o n t a i n m a i n l y t h e l i g h t e r c h l o r o p l a s t DNA. T h e i n t e r m e d i a t e f r a c t i o n (d) is a m i x t u r e of h e a v y a n d l i g h t DNA. The DNA samples were characterized by their denaturation-renaturation behavior. The results a r e g i v e n i n figure 9. P a t t e r n s b, c, d, a n d e b e l o n g to t h e d e n a t u r e d D N A ; p a t t e r n s b', c', d', a n d e b e l o n g to t h e r e n a t u r e d DNA. All d e n a t u r e d D N A s a m p l e s s h o w t h e t y p i c a l i n c r e a s e in b u o y a n t d e n s i t y of a p p r o x i m a t e l y 15 rag. T h e s a m p l e s differ, h o w e v e r , in t h e i r r e n a t u r a t i o n b e h a v i o r s . As exp e c t e d [28], t h e c h l o r o p l a s t D N A (e, e') r e g a i n s
Hydroxyapatite c h r o m a t o g r a p h y of E u g l e n a DNA. almost c o m p l e t e l y the density of the native DNA (1.687 versus 1.185 g/cm3). The n u c l e a r DNA (b, b') s h o w e d after r e n a t u r a t i o n a b r o a d p e a k w i t h a m e a n b u o y a n t d e n s i t y of 1.718 g/cm3. The profile of the t w o i n t e r m e d i a t e f r a c t i o n s reveal t w o
I I I
b
F i g u r e 10 s h o w s the c h r o m a t o g r a m o b t a i n e d w i t h the s e c o n d c h l o r o p l a s t DNA p r e p a r a t i o n . As a l r e a d y m e n t i o n e d , in this case (not shown) nuclear DNA (1.707 g/cm3) w a s not detectable, but the satellite DNA (1.700 g / c m 3) w a s v e r y p r o m i nent. F i g u r e 11 s h o w s the b u o y a n t d e n s i t y profiles o b t a i n e d on p o o l e d f r a c t i o n s f r o m the chrom a t o g r a m . It is e v i d e n t that f r a c t i o n s eluttng at i n c r e a s i n g l y h i g h e r m o l a r i t i e s b e c o m e e n r i c h e d in c h l o r o p l a s t DNA w i t h c o n c o m i t a n t d e c r e a s e of satellite DNA. I
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I
1.685 1.699
I
I I 1.742 Fro. 11. - - Scannings of pooled DNA fractions from the chromatogram of figure 10. a) 52-59 ; b) 60-61 : c) 6,2-65 ; d) 66-73. Centrifugation conditions in neutral CsCl density gradient as gi'ven in figure 2.
Fro. 12. - - Preparative CsC1 density gradient of (a) satellite DNA (2.28 A~o units) (b) ehloroplast DNA (0.94 A~o units). The DN,A preparations used in this experiment were obtained from the pooled a + b and c + d fractions of the chromatogram shown in figure 10, respectively. Divisions on the abseissa scales correspond to 1.93 ml. Arrows indicate the central fractions which were used for analytical gradient (fig. 13) and base analysis (table I). ¢1
c o m p o n e n t s after r e n a t u r a t i o n s . Profile c' s h o w s a s h a r p p e a k w i t h a d e n s i t y of 1.703 g / c m a and a b r o a d e r c o m p o n e n t s w i t h an a v e r a g e density of 1.714 g / c m 3. The sample d' contains p r o b a b l y some c h l o r o p l a s t DNA (1.688 g/cm3) and a comp o n e n t of density 1.700. g / c m 3, p r o b a b l y i d e n l i fiable w i t h satellite DNA r e c e n t l y d e s c r i b e d [17]. The results of figure 9 suggest that the satellite !)NA is eluted f r o m h y d r o x y a p a t i t e at an i n t e r m e diate p o s i t i o n b e t w e e n n u c l e a r and c h l o r o p l a s t DNA, a c o n c l u s i o n c o n f i r m e d by the f o l l o w i n g experiments. TABLE 1.
Nucleoside analysis o[ chloroplast and satellite DNA's. I G i__
T
C
/', A
G + C
- - i - -
I Chloroplast DNA(a)
12.912.9
37.8 37.4
12 1 !i 37.2 12.2 [ 37.5
25.0 25,1
Satellite DNA (b)
2122.76
28.6 28.9
19.2 19,2
41.8 40.9
29.5 30.2
(u) Duplicate analysis on Bio Gel-2 coluinn. (b) Duplicate analysis on Aminex A6 column. BIOCHIMIE, 19,72, 54, n ° 8.
I
1.684 I 1.742 1.700 FiG. 13. - - Analytical GsG1 density gradient of (a) satellite DNA (b) chloroplast DNA. The DNA preparations used in this experiment originated from the centra.1 fractions of the preparative gradients shown in the previous figure.
F r a c t i o n s a + b and c + d f r o m the c h r o m a t o g r a m of figure 10 w e r e f u r t h e r purified by p r e p a r a t i v e CsCl density g r a d i e n t s (fig. 12). F i g u r e 13 s h o w s the a n a l y t i c a l s c a n n i n g s of these p u r i f i e d fractions, c o r r e s p o n d i n g to satellite (~ = 1.700 g/ era3), a n d c h l o r o p l a s t (~ -- 1.785 g/cm3) DNA. N u c l e s i d e analysis for these p r o d u c t s are s h o w n in table I. T h e GC levels found f o r c h l o r o p l a s t and
E. S t u t z a n d G. Bernardi.
1020
s a t e l l i t e D N A w e r e 25 p. c e n t a n d 40 p. c e n t , r e s pectively, in good agreement with the buoyant d e n s i t y r e s u l t s . T h e s e d i m e n t a t i o n c o e f f i c i e n t s of t h e t w o D N A ' s w e r e 218S a n d 28 S, r e s p e c t i v e l y .
DISCUSSION.
Acknowledgments. The Euglena c h l o r o p l a s t a n d m i t o c h o n d r i a l DNA samples were p r e p a r e d b y Mr. J. P. V a n d r e y a n d Mr. E. J. Crouse, N o r t h w e s t e r n U n i v e r s i t y ; t h e technical assistance of Miss C. G a i l l a r d is graCefully acknowledged. This w o r k w a s supported, i n part, b y g r a n t GB 19191 from the N a t i o n a l Science F n u n d a t i o n a n d a special Research G r a n t f r o m N o r t h w e s t e r n U n i v e r s i t y to one of u s (E.S.).
The present work establishes chromatography o n h y d r o x y a p a t i t e as a n e w t o o l f o r t h e p r e p a r a t i o n of b o t h m i t o c h o n d r i a l a n d c h l o r o p l a s t D N A ' s f r o m Euglena gracilis. I n a d d i t i o n , a s a t e l l i t e DN,A h a v i n g a b u o y a n t d e n s i t y of 1.700 g / c m 3 c a n also be separated from chloroplast-enriched preparat i o n s . T h e c e l l u l a r o r i g i n of t h i s s a t e l l i t e D N A will be discussed elsewhere (paper in preparation).
Nous avons gtudi6 ]e c o m p o r t e m e n t e h r o m a t o g r a p h i q u e des DNAs n a t i f s d'Euglena gracilis sur eolonnes d ' h y d r o x y a p a t i t e . Des p r g p a r a t i o n s d'acides nuclgiques t o t a u x peuvent ~tre fractionn~es sur colonne, en DNA nucIdaire et DNA des o r g a n i t e s ( m i t o e h o n d r i e s e.t chloroplastes). Une s~paration en <> p e r m e t d'obten i r r a p i d e m e n t le DNA des o~ganites ~t p a r t i r de pr~para¢ions d?aeides uuel~iques; totaux.
T h e p r a c t i c a l a d v a n t a g e s of t h e h y d r o x y a p a t i t e chromatography, particularly in its batchwise oper a t i o n , h a v e b e e n d i s c u s s e d e l s e w h e r e El0] a n d are too obvious, especially for large-scale preparations, to be further commented upon here.
La c h r o m a t o g r a p h i c de p r 6 p a r a t i o n s de DNA de m u t a n t s 6tiol6s p a r la c h a l e u r p e r m e t de s6parer le DNA m i t o c h o n d r i a l du DNA nucl6aire. La c h r o m a t o graphic de p r g p a r a t i o n s de DNA o b t e n u e s h p a r t i r de f r a c t i o u s d'Eugl~ne enrichies en chlorolalastes p e r m e t de prdparer le D NA des cbioroplastes et, en outre, u n DNA satellite d~jh d~crit (p ---- 1.7,00, g/cm~).
I t m a y b e i n t e r e s t i n g to r e m a r k t h a t t h e f o u r D N A ' s f r o m Euglena i n v e s t i g a t e d h e r e , n a m e l y t h e n u c l e a r DNA, t h e s a t e l l i t e D N A (? = 1.70.0 g / e m a ) , mitoehondrial DNA and chloroplast DNA are evidently eluted in the order of increasing AT cont e n t s . S o m e s a t e l l i t e D N A ' s also a p p e a r to b e e l u t e d a c c o r d i n g to t h e i r A T c o n t e n t s : f o r i n s t a n c e , s a t e l lite I a n d I I f r o m c a l f t h y m u s , w h i c h a r e r i c h e r i n GC c o m p a r e d w i t h m a i n b a n d DNA, a r e e l u t e d at a l o w e r m o l a r i t y t h a n t h e l a t t e r [5-7] ; m o u s e s a t e l l i t e w h i c h is p o o r e r i n GC c o m p a r e d w i t h m a i n b a n d D N A is e l u t e d a f t e r t h e l a t t e r [6, 7]. I t m i g h t b e t e m p t i n g to c o n c l u d e t h e r e f o r e t h a t A T - r i c h D N A ' s a r e e l u t e d f r o m h y d r o x y a p a t i t e at h i g h e r m o l a r i t i e s t h a n G C - r i e h D N A s, as t h e y a r e from methylated serum albumin - kieselguhr c o l u m n s [29], p a r t i c u l a r l y i n v i e w of t h e d i f f e r e n t secondary structure reported or suggested for very A T - r i c h D N A ' s [30, 31]. S u c h a c o n c l u s i o n i n all l i k e l i n e s s is n o t of g e n e r a l v a l i d i t y s i n c e t h e elut i o n m o l a r i t i e s of n a t i v e D N A ' s f r o m h y d r o x y a p a t i t e c o l u m n s a r e p r o b a b l y r e l a t e d t o s t r u c t u r a l features which do not depend upon the overall base c o m p o s i t i o n i n a s i m p l e w a y . I t is p o s s i b l e , h o w e ver, that the high elution molarity from hydroxya p a t i t e is a p r o p e r t y c o m m o n to m a n y A T - r i c h D N A ' s a n d r e f l e c t s t h e p r e s e n c e i n t h e s e D~NA's of short alternating and non-alternating AT sequences ; i n v e s t i g a t i o n s of o t h e r A T - r i c h D N A ' s s u c h as t h e m i t o c h o n d r i a l D N A ' s f r o m Tetrahgmena pgriformis [32] o r Physarum polgcephalum [33] might be interesting in this connection.
BIOCHIM1E, 19.72, 54, n ° 8.
Rgsu~.
Nous pr6seutons ici quelques propri4t~s p h y s i q u e s et c h i m i q u e s des; DNAs d'Eugl6ne o b t e n u s p a r c h r o m a tographic sur h y d r o x y a p a t i t e . ZUSAMMENFASSUNG. W i r h a b e n das B e n e h m e n der n a t i v e n DNA yon
Euglena gracilis an H y d r o x y a p a t i t s / i u l e n nnters.ucht. Pr~iparate von g e s a m t e n N,ukleins~iuren k 6 n n e n a n einer S~iule in Nukleus- u n d Organellen (Mitoehondrien u n d Chloroplasten)~-DNA f r a k t i o n i e r t werden. Eine <>-Trennung e r l a u b t es die Organellen-DNA aus Pr~iparaten yon g e s a m t e n Nukleinsfiuren rasch zu erhalten. Die C h r o m a t o g r a p h i c yon P r i i p a r a t e n der DNA a~s d u r c h Hitze v e r b l a s s t e n M u t a n t e n e r l a u b t die Trenn u n g der Mitochondrien-DNA yon der Nukleus-DNA. Die C h r o m a t o g r a p h i c tier aus. E u g l e n f r a k i i o n e n , welche an Chlorop~asten a n g e r e i e h e r t Wurden, e r h a l t e n e n DNA-Pr~iparate e r l a u h t es die Chloroplasten-DNA zu e r h a l t e n u n d a u s s e r d e m eine schon b e s c h r i e b e n e Satellit-DNA (9 : 1 700 g/cma). W i r stellen h i e r einige p h y s i k a l i s e h e n u u d chemist h e E i g e n s c h a f t e n der Euglen-DNA vor, w.elche d u r c h Chromatographic an Hydroxyapatit erhalten wurden.
REFERENCES. 1. Bernardi, G. (1962) Biochem. J., 83, 32r p. 2. Bernardi, G. (1965) Nature, 206, 779,. 3. Bernardi, G. (1969~ Biochim. Biophys. Acta, 174, 423. 4. Bernardi, G. (1971) Methods in Enzymology. L. Gross~lan a n d K. Moldave eds. Academic Press, New York, vol. 21, p. 95. Bernardi, G. (1971) Procedures in Nucleic Acids Research. G. L. Cantoni a n d D. R. Davies eds. H a r p e r et Row, New York, vol. 2, p. 445. 5. Andre, M. (1971) Thesis (Strasbourg).
Hydroxyapatite
chromatography
6. Filipski, J., Thiery, J. P. a Bernardi, G. in p r e p a r a tion. 7. Corneo, G., Zardi, L. ~, Polli, E. (1970) Biochim. Biophys. Acta, 217, 249. 8. Bernardi, G., Carnevali, F., Nicolaieff, A., Piperno, G. a Tecce, G. (19'68) J. Mol. Biol., 37, ~9~3. 9. Bernardi, G., Faurbs, M., Piperno, G. ~ Slonimski, P. (197'0) J. Mol. Biol., 48, 23. 10. Bernardi, G., Piperno, G. ~ Foniy, G. (19.72) J. MoL Biol., 65, 173. 11. Bcrnardi, G. & Timasheff, S. N. (1970) J. Mol. Biol., 48, 43. 12. Piperno, G., Fonty, G. & Bernardi G. (1972) J. Mol. Biol., 65, 191. 13. Prunell, A. a Bernardi, G. in preparation. 14. Schiff, J. A. (1970) Syrup. Soc. Explt. Biol., 24, 277. 15. Schiff, J. A. (1971) in Autonomy and Biogenesis of Mitochondria and Chloroplasts, B o a r d m a n N. J., Linnane A. W., Smillie R. M., (eds.), North Holland, A m s t e r d a m , p. 277. 16. Ra~wson, J. R. ,¢ Stutz, E. (196'9) Biochim. Biophys. Acta, 190, 36~8. 17. Stutz, E. ~ Vandrey, J. P. (19,71) Febs Letters, 17, 277. 18. Kranviec, S. ~ Eisenstadt, J. M. (1~970) Biochim. Biophys. Acta, 217, 12'0. 19. Mandel, J. D. ~ Hershey, A. D. (1960) Anal. Biochem., 1, 66.
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DNA.
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20. Piperno, G. ~ Bernardi, G. (1971) Biochim. Biophys. Acla, 238, 388. 21. Thiery, J. P., Ehr.lich, S. D. ~ Bernardi G. Biochemistry, submitted for publication. 22. B r a w e r m a n , G. (1968) in the Biology of Euglena ; D. E. Buetow (ed.), Academic Press, New York, p. 97. 23. Edelman, M., Cowan, C. A., Epstein, H. T. & Schiff, J. A. (1964) Proc. Natl. Acad. Sci., 52, 1214. 24. B r a w e r m a n , G. ~ Eisenstadt, J. M. (1964) Biochim. Biophys. Acta, 91. 477. 25. Ray, S. D. ~ Hanavealt, P. C. (19:64) J. Mol. Biol., 9, 812. 26. Ray, S. D. & Hanawa,lt, P. C. (1965) J. Mol. Biol., 11, 760. 27. Edelman, M., Schiff, J. A. ~ Epstein, H. T. (1965) J. Mol. Biol., 11, 769. 28. Stutz, E. ~ Rawson, J. R. (lff70) Biochim. Biophys. Acta, 209, 16. 29. Sueoka, N. ~ Cheng, T. Y. (1963) J. Mol. Biol., 4, 1'61. 30. Bram, S. (19'71) Nature, N e w Biology, 232, 174. 31. Pilet J. a Brahms, J. (1972) Nature, New Biology, 236, 99. 32. Suyama, Y. (1966) Biochem., 5, 2214. 33. Evans, T. E. (19'66) Biochem. Biophys. Res. Commun., 22, 678.
69
Hydroxyapatite chromatography of deoxyribonucleic acids from Euglena gracilis. E. STUTZ and G. BERNARDI.
Laboratoire de G~t~tique Moldct:laire, Institut de Bioloyie Moldculaire, Paris 5", Fral~ce and Department o[ Bioloyical Sciences, Northweslern University, Evanston, Ill. 60 201. (3/6/1972). Summary. -- The chromatographic behavior of native Euglena 9racilis DNA's on hydroxya,patite columns has been investigated. Total nucleic acid preparations can be f'ractionated by column chromatography to yield nuclear DNA and organeHe (mitoehondrial + chloroplast) DNA. A batch procedure permits the rapid separation of organeHe DNA from ,total nucleic acid preparations. Chromatography of DNA preparations from heat-bleached mutants permits the separation of mitochondrial DNA from nuclear DNA. Chromatography of DNA preparations from chloroplast-enriched fractions of Euglena permits the separation of ch,loroplast DNA, and in addition, of a satellite DNA previously described (~ = 1.700 g/emS). Some physical and chemical properties of ~he Euglena DNA's, as obtained by chromatography on hydroxyapatite, are reported.
INTRODUCTION. T h o u g h mostly used for the s e p a r a t i o n of native a n d d e n a t u r e d DNA [1, 2], h y d r o x y a p a t i t e (HA) c o l u m n s have a c o n s i d e r a b l e d i s c r i m i n a t o r y p o w e r t o w a r d s different native DNA structures [3, 4q. Not t a k i n g into c o n s i d e r a t i o n the case i n w h i c h the s e c o n d a r y (single-stranded DNA from ¢ X 174 phage) or the t e r t i a r y structure (twisted c i r c u l a r DNA from p o l y o m a virus) are grossly different and cause a different c h r o m a t o g r a p h i c behavior [3, 4], it is b e c o m i n g i n c r e a s i n g l y evident that differences i n nucleotide sequences i n otherwise s i m i l a r DNA's (the s i m i l a r i t y c o n c e r n ing the double-strandedness, the m o l e c u l a r weight, the linear, or open circular, configuration, and also the n u c l e o t i d e composition) m a y be sufficient to d e t e r m i n e different elution molarities. Recent w o r k [5-71 suggests that DNA's c o n t a i n i n g short repetitive sequences like satellite DNA's may, in general, show p a r t i c u l a r elution molarities. A p a r t i c u l a r l y s t r i k i n g case is that of m i t o c h o n drial DNA's from wild-type a n d cytoplasmic (
elution molarities of yeast m i t o c h o n d r i a l DNA's are due to the presence i n these DNA's of long AT-rich stretches [10] c o n t a i n i n g both a l t e r n a t i n g dAT : dAT and n o n - a l t e r n a t i n g dA : dT s t r u c t u r e s [11]. I n fact, poly ( d A T : dAT) a n d poly ( d A : dT) a n d the dAT-rich satellite DNA from Cancer pagurus, elute at slightly a n d m u c h higher molatitles, respectively, than bacterial DNA's [10] ; finally e x p e r i m e n t s on e n z y m a t i c a l l y degraded yeast m i t o c h o n d r i a l DNA's have s h o w n that GCrich fragments elute at a l o w e r phosphate molarity compared to the AT-rich fragments [12, 13]. The results with yeast m i t o c h o n d r i a l DNA p r o m p t e d a s i m i l a r k i n d of e x p e r i m e n t s with Euglena qracilis chloroplast a n d m i t o c h o n d r i a l DNA w h i c h both are low in their GC contents (24 p. cent a n d 32 p. cent, respectively) [14]. These DNA's might share some s t r u c t u r a l features w i t h the m i t o c h o n d r i a l DNA's from yeast a n d display, therefore, a s i m i l a r c h r o m a t o g r a p h i c behavior. F r o m a p r a c t i c a l p o i n t of view, tile s e p a r a t i o n of organellar from n u c l e a r DNA i n the case of Euglen.a is complicated by the fact that the former only represents 1-2 p. cent of the total cellular DNA, a level about 10 times lower t h a n that of yeast n l i t o c b o n d r i a l DNA. If successful, however, the method w o u l d lend itself to scale tip the prep a r a t i o n of o r g a n e l l a r DNA in a relatively simple a n d i n e x p e n s i v e way, a p o i n t of c o n s i d e r a b l e im-
E. Stutz and G. Bernardi.
1014
p o r t a n e e in v i e w of the c u r r e n t i n t e r e s t in the b i o l o g i c a l f u n c t i o n of b o t h DNA's [151. In the f o l l o w i n g w e r e p o r t the c h r o m a t o g r a p h i c s e p a r a t i o n of b o t h Euglena c h l o r o p l a s t a n d mitoc h o n d r i a l DNA u s i n g e i t h e r total c e l l u l a r n u c l e i c a c i d s or o r g a n e l l a r DNA's e n r i c h e d s a m p l e s as s t a r t i n g materials. In a d d i t i o n , the p r o c e d u r e used p e r m i t s the s e p a r a t i o n of n u c l e a r DNA and of a satellite DNA. This w o r k has been p r e s e n t e d at the Colloque de la Soci6t6 de C h i m i e Biologique sur les Acides D ~ s o x y r i b o n u c l ~ i q u e s des E u c a r y o t e s (Strasbourg, March 16-18, 1972). MATEI~IALS AND METHODS. 1) Cultivation of cells. Euglena gracilis, Klebs (Z-strain ; Culture Coll e c t i o n of Algae, I n d i a n a U n i v e r s i t y , N ° 753) w a s g r o w n a u t o t r o p h i c a l l y , h a r v e s t e d a n d s t o r e d as r e p o r t e d e a r l i e r [16]. Euglena gracilis, heat-bleac h e d mutant, w a s g r o w n h e t e r o t r o p h i c a l l y on Euglena B r o t h (Difco) either in light o r in d a r k . The m u t a n t was i s o l a t e d in E v a n s t o n a n d g r o w n for m o r e t h a n 12 m o n t h s w i t h o u t s h o w i n g a n y green i n g c a p a c i t y . No c h l o r o p l a s t DNA (0 = 1.685 g/ c m 3) could be d e t e c t e d in this mutant. 2) Parification of chloroplast and mitochondria. C h l o r o p l a s t s w e r e i s o l a t e d f r o m w i l d - t y p e cells as r e p o r t e d [16], a n d p u r i f i e d b y flotation in a L u d o x g r a d i e n t [17]. M i t o c h o n d r i a w e r e i s o l a t e d f r o m b l e a c h e d cells w h i c h w e r e b r o k e n u s i n g a F r e n c h P r e s s at 2 000 psi, in 0.25 M sucrose, 0.01 M tris HC1, p H 7.9, 0.25 M KC1, 0.00,4 M MgCl 2 a n d 0.005 M ~ - m e r c a p t o e t h a n o l . The c r u d e mitoc h o n d r i a w e r e o b t a i n e d b y d i f f e r e n t i a l centrifugation a c c o r d i n g to K r a w i e c a n d E i s e n s t a d t [181. 3) Nucleic acid extraction. Total n u c l e i c acid. 60 g (wet p a c k e d ) w i l d t y p e cells w e r e r e s u s p e n d e d in 0.15 M NaC1 cont a i n i n g 2.5 p. cent s o d i u m d o d e c y l s u l f a t e (SDS) (150 ml). The m i x t u r e w a s a d j u s t e d to 1 M NaCl w i t h NaC1 c r y s t a l s and s t i r r e d o v e r n i g h t at 4°C. The s u s p e n s i o n w a s c e n t r i f u g e d for 20 rain at 27,000 × g (Sorvall SS-34) a n d the s u p e r n a t a n t p r e c i p i t a t e d w i t h 1 volume of c o l d ethanol. The p r e c i p i t a t e was r e c o v e r e d b y s p i n n i n g at 27,000 × g, for 20~ m i n (Sorvall SS-34), r e d i s s o l v e d in 0.01 M NaC1 (50 ml), a d j u s t e d to 1.5 p. cent SDS, 0.15 M NaC1 a n d s t i r r e d for 30 rain. After adiust e m e n t of NaC1 to 1 M and r e p e t i t i o n of s t i r r i n g o v e r n i g h t at 4°C, the s u s p e n s i o n w a s again centrifuged at 27,000 × g, for 20 rain a n d the s u p e r n a t a n t p r e c i p i t a t e d w i t h 1 volume of ethanol. The
BIOCHIM1E, 1972, 54, n ° 8.
r e c o v e r e d p r e c i p i t a t e w a s dissolved in 0.15 M NaCI (15 ml) a n d the solution e x t r a c t e d w i t h chlor o f o r m - i s o a m y l alcohol ( 2 4 : 1 ; v/v). The chlorof o r m e x t r a c t i o n was r e p e a t e d 3 times a n d the final aqueous p h a s e w a s d i a l y z e d against 0.01 M s o d i u m p h o s p h a t e buffer a n d used for h y d r o x y a p a t i t e c h r o m a t o g r a p h y (total A260 units : 10'21). In some eases the n u c l e i c a c i d e x t r a c t was t r e a t e d w i t h r i b o n u c l e a s e as specified b e l o w (Section 4). 4) DNA from chloroplasts and mitochondria. The p u r i f i e d organelles from 50 g of e i t h e r green or b l e a c h e d cells w e r e r e s u s p e n d e d in 10 ml of 0.1 M tris-HCl (pH 7.5), 2.5 p. cent SDS, 0.01 M EDTA, a n d h e a t e d for 10 rain to 60 °. The suspension w a s a d j u s t e d to 1 M NaC104 a n d e x t r a c t e d t w i c e w i t h 2 volumes of c h l o r o f o r m - i s o a m y l alcohol ( 2 4 : 1 ; v/v). The final aqueous l a y e r w a s digested w i t h BNAase ( r i b o n u c l e a s e B, bovine p a n creas, t y p e VII, Sigma) r e e x t r a e t e d a n d d i a l y z e d o v e r n i g h t against 0.1 M NaC1-0.05 M s o d i v m phosp h a t e buffer, p H 6.8, 0.1 M EDTA. The d i a l y z a t e was p u r i f i e d on a m e t h y l a t e d s e r m n a l b u m i n k i e s e l g u h r (MAK) [191 column. The DNA p e a k w a s p o o l e d a n d d i a l y z e d against 0.1 × s t a n d a r d saline c i t r a t e (SSC ; 0..15 M NaC1, 0.0,15 M citrate, p H 7.5). 1.2 to t.5 A2s0 units w e r e r e c o v e r e d f r o m green c e l l s ; 10-15 A2so units f r o m b l e a c h e d mutant. The degree of c o n t a m i n a t i o n of c h l o r o p l a s t DNA b y n u c l e a r DNA v a r i e d from p r a c t i c a l l y n o t h i n g to about 60 p. cent. The m i t o c h o n d r i a l D'NA p r e p a r a t i o n used h e r e c o n t a i n e d 90 to 95 p. cent of n u c l e a r DNA. 5) Chromatography on hydroxgapatite. Column a n d b a t c h p r o c e d u r e s w e r e p a t t e r n e d after the e x p e r i m e n t s d e s c r i b e d e l s e w h e r e on y e a s t m i t o c h o n d r i a l DNA [10]. 6) Ultracentrifugation experiments. S e d i m e n t a t i o n v e l o c i t y e x p e r i m e n t s a n d analytical caesium chloride density-gradient experim e n t s on native a n d d e n a t u r e d DNA w e r e done as d e s c r i b e d e l s e w h e r e El0]. P r e p a r a t i v e c a e s i u m c h l o r i d e d e n s i t y g r a d i e n t e x p e r i m e n t s w e r e also p e r f o r m e d as d e s c r i b e d e l s e w h e r e [61. Denaturation- renaturation experiments were p e r f o r m e d as follows : DNA s a m p l e s (2-3 ~Jg in 0.5 ml) in 0.05 M NaC1 w e r e d e n a t u r e d b y h e a t i n g at 100 ° for 10 min, f o l l o w e d b y q u e n c h i n g in an ice bath. D e n a t u r e d s a m p l e s w e r e r e n a t u r e d b y a d j u s t i n g the solvent to 2 × SSC w i t h 10 × SSC a n d b y k e e p i n g t h e m f o r 4 h o u r s at 64 °. 7) Base composition of DNA. This w a s d e t e r m i n e d b y e n z y m i c a l l y d e g r a d i n g DNA to n u c l e o s i d e s and b y a n a l y z i n g the l a t t e r
Hydroxyapatite chromatography of E u g l e n a o n e i t h e r B i o Gel P-2 c o l u m n s a c c o r d i n g to P i p e r n o a n d B e r n a r d i [20] o r A m i n e x A6 c o l u m n s as d e s c r i b e d b y T h i e r y , E h r l i c h a n d B e r n a r d i [21].
DNA.
1015
bed, except that no RNA peak was evident ; some low-eluting material, probably formed by residual, d e g r a d e d RNA, w a s f o u n d i n t h e s e p r e p a r a t i o n s .
0.8 =L F
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El
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06
./1///IJ~t/1
0.3 0.2
0.4
0.1 ~_ oc
0.2
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0 0
50
I O0
150
FIG. 1. - - C h r o m a t o g r a p h y of a t o t a l nucleic acid extract of Euglena gracilis. 150 ml of nucleic acid solution in 0.01 M sodium p h o s p h a t e buffer (150 A~0 units) were loaded on a 1.08 × 60 cm h y d r o x y a p a t i t e column e q u i l i b r a t e d w i t h t h e same buffer. 1'00 ml of this buffer were used to w a s h the column and a 0.01 to 0.5 M g r a d i e n t of sodium p h o s p h a t e buffer, pH 6.8 (1200 ml) w a s applied. 3.4 ml fraction were collected. Flow rate was 34 re,l/hr. Circles indicate 'the l i m i t s of the f r a c t i o n s w h i c h were pooled together.
200
FRACTION NUMBER
RESULTS. Figure 1 shows the hydroxyapatite chromatog r a m of a t o t a l n u c l e i c a c i d e x t r a c t f r o m Eufflena yracilis. T h e f i r s t b i p h a s i e p e a k is f o r m e d b y R N A w h i c h is w e l l s e p a r a t e d f r o m t h e s e c o n d m a j o r p e a k r e p r e s e n t i n g DNA. 2,1.7 A~60 u n i t s w e r e r e c o v e r e d u n d e r t h e D N A p e a k , w h i c h is a p p r o x i m a t e l y 15 p. c e n t of t h e t o t a l A~60 i n p u t . T h i s r o u g h l y c o r r e s p o n d s to t h e D NA : R N A r a t i o f o u n d i n Euglena w i l d - t y p e c e l l s [23J. T h e b u l k of t h e D N A e l u t e d at a p h o s p h a t e c o n c e n t r a t i o n of 0.28 M. T h e fractions under the DNA peak were pooled in four D N A s a m p l e s a, b, c, d, w h i c b , a f t e r c o n c e n t r a t i o n and dialysis, were analyzed for their buoyant dens i t i e s i n n e u t r a l CsCA. T h e f o u r p r o f i l e s a r e s h o w n in figure 2 and though the gradients were overl o a d e d i n c a s e a, b, a n d c, n o s a t e l l i t e D N A w a s detectable. It was only in sample d that a compon e n t of l o w e r b u o y a n t d e n s i t y (~ = 1.6,85 g/cm,~) s h o w e d u p i n a d d i t i o n to t h e m a i n n u c l e a r D N A (~ ~ 1.707 g / c m 3 ) . T h e 1.685 g / c m a b u o y a n t d e n s i t y is t y p i c a l f o r Euglena c h l o r o p l a s t D N A [23, 24, 25]. T h e m i t o c h o n d r i a l D N A w i t h a r e p o r t e d d e n s i t y of 1.690 g / c m a [26, 27] c o u l d n o t b e disc e r n e d i n a n y of t h e s e p r o f i l e s . T h e s a t e l l i t e p e a k r e p r e s e n t e d 15 p. c e n t of t h e n u c l e a r p e a k i n p r o file d a n d s o m e w h a t l e s s t h a n 1 p. c e n t of t h e t o t a l DNA. T h e c h r o m a t o g r a p h i c b e h a v i o r of t o t a l n u c l e i c acid preparations which had undergone RNase t r e a t m e n t w a s e x a c t l y t h e s a m e as t h a t just d e s c r i -
BIOCHIMIE, 19:72, 54, n o 8.
T h e f a c t t h a t t h e o r g a n e l l e D N A is e l u t e d at t h e highest molarity in the chromatogram is v e r y
I I
i I
i
I
I
1.685 I 1.707 I t.742 FiG. 2. - - Seannings of pooled DNA fractions f r o m the h y d r o x y a p a t i t e c h r o m a t o g r a m of figure 1. a) 147-156 (280 nm) ; b) 157-181 (28[} n m ) ; c) 182190 (280 nm) ; d) 191-220. Samples were centrifuged at 44,000 r e v / m i n for 20 h r at 25°C in a n e u t r a l Cs'CI density gradient. DNA from phage 2 C (1.742 g/cm~) was used as a density marker. Scannings were done at 265 nm, u n l e s s otherwise indicated by a n u m b e r in brackets a f t e r the fraction number.
E. S t u t z a n d G. B e r n a r d i .
1016
f a v o r a b l e for i s o l a t i n g this c o m p o n e n t u s i n g a b a t c h p r o c e d u r e [10]. K n o w i n g f r o m the first exp e r i m e n t that the bulk of n u c l e a r DNA was eluted w i t h 0.28 M s o d i m n p h o s p h a t e buffer, 50 ml of hyd r o x y a p a t i t e , p r e v i o u s l y e q u i l i b r a t e d w i t h 0.28 M p h o s p h a t e , w e r e s u s p e n d e d in a total v o l u m e of 100 ml of 0.28 M p h o s p h a t e c o n t a i n i n g 871 A ~ 0 units of total n u c l e i c acid. The suspension was gently s t i r r e d o v e r n i g h t at r o o m t e m p e r a t u r e . T h e HA crystals w e r e w a s h e d t h r e e times w i t h 50 mlv o l u m e s of 0.28 M p h o s p h a t e and l a y e r e d on a 2 × 60 cm h y d r o x y a p a t i t e c o l u m n e q u i l i b r a t e d w i t h the same buffer. T h e c o l u m n was w a s h e d w i t h 80 ml of 0.28 M buffer and eluted w i t h a l i n e a r 0.28 to 0..48 M (1,920 nil) m o l a r i t y g r a d i e n t of phosphate. The flow rate was 6.8.5 m l / h . F r a c tions of 5.7 nfl w e r e collected. U n d e r these conditions, 3.25 A.260 units w e r e eluted ( c h r o m a t o g r a m not s h o w n ) . T h r e e DNA samples w e r e o b t a i n e d by p o o l i n g f r a c t i o n s eluted b e t w e e n the f o l l o w i n g p h o s p h a t e m o l a r i t i e s : a, 0.28-0.29 ; b, 0.29-0.30 ; c, 0.30-0.32. T h e r e c o v e r y in a, b, c w e r e 1.0,7, 0.62, 1.56, A260 units, r e s p e c t i v e l y . T h e p o o l e d f r a c t i o n s w e r e c o n c e n t r a t e d by r o t a r y e v a p o r a t i o n , d i a l y z e d
5-10 p. cent, and 70-80 p. cent, r e s p e c t i v e l y . The a m o u n t of this l i g h t e r DNA, c o r r e s p o n d i n g to organelle DNA (see below), e x t r a c t e d by the b a t c h m e t h o d is e q u i v a l e n t to the a m o u n t of organelle DNA f o u n d in the c h r o m a t o g r a m of figure 1. This i n d i c a t e s that the b a t c h m e t h o d gives the same y i e l d as c o l u m n c h r o m a t o g r a p h y , and is in essence feasible for large-scale p r o d u c t i o n of o r g a n e l l a r DNA f r o m Euglena. The p u r i f i c a t i o n of c h l o r o p l a s t DNA f r o m Eaglena cells is c o m p l i c a t e d by the p r e s e n c e of mitoc h o n d r i a l DNA, since the f r a c t i o n eluting at high m o l a r i t y m i g h t c o n t a i n the m i t o c h o n d r i a l DNAas
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FIG. 4. Chromatography of DNA from a mitoehondria-enriched fraction from a bleached mutant of Euglena gracilis. 25 inl of DNA solution in 0.1 M sodium phosphate buffer (2,5.2 A~o units) were loaded on a 1.08 × 60, cm hydroxyapatite column equilibrated with the same buffer. The c(~lumn was washed with 30 ml of O]is buffer and a gradient of 0.1 to 0.5 M sodium phosphate (80,0 lnl) was applied. 4.5 ml fractions were collected. Flow rate Was 28 ml/h. A~eorecovery was 92 p. cent. Circles, indicate the limits of the fractions which were pooled together. -
I
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1.7o7 I ( 1.742 Fro. 3. - - Seannings of pooled DNA fractions from t h e batch experiment (see Tex,t). a) 40-54 ; b) 55.-68 ; c) 69,-92. The mo]ari.ty of the sodium 'phosphate buffer were 0.28 at fraction 40, 0.29 at fraction 55, 0,30 a tfraction 69,. Gentrifugation conditions in neutral CsCil density gradients as given in figure 2.
against 0.1 M NaCJ a n d aliquots w e r e a n a l y z e d by u l t r a c e n t r i f u g a t i o n in CsC1 density gradients (fig. 3). Sample a c o n t a i n e d only n u c l e a r DNA (1.707 g / c m 3) ; sample b and c c o n t a i n e d , in addition, a l i g h t e r DNA (1.68,5 g / c m 3) at levels of about
BIOCHTMIE, 19.72, 54, n ° 8.
well. T h e profile e (,fig. 3) dose not r e v e a l any c o m p o n e n t at 1.6,90 g f c m 3, w h i c h is the r e p o r t e d b u o y a n t density of Euglena m i t o c h o n d r i a l DNA. T h e r e f o r e , e i t h e r the m i t o c h o n d r i a l DNA eluted at a different m o l a r i t y or the two satellites did not b a n d sufficiently apart. To clarify this point, w e c h r o m a t o g r a p h e d DNA extracted from a mitochondria-enriched fraction o b t a i n e d fronl a h e a t - b l e a c h e d mutant. F i g u r e 4 shows the c h r o m a t o g r a m w h i c h was again separated into four fractions, a, b, c a n d d. The d e n s i t y profiles of these f r a c t i o n s are s h o w n in figure 5. Again, it is only the last f r a c t i o n (buffer c o n c e n t r a t i o n 0.3'0-0.35 M) w h i c h contains the satellite, thougb still h e a v i l y c o n t a m i n a t e d w i t h n u c l e a r DNA. The b u o y a n t d e n s i t y in neutral CsC1 of the m i t o c h o n d r i a l DNA was 1.6,88 g/em3, slightly
Hydroxgapatite chromatography of b e l o w t h e r e p o r t e d v a l u e s [26, 27]. I d e n t i c a l d e n s i t y v a l u e s (1.688 g / e r a 3) w e r e o b t a i n e d , h o w e v e r , for mitochondrial DNA isolated from a bleached m u t a n t [18]. T h e p r o x i m i t y of t h e d e n s i t i e s of chloroplast and mitochondrial DNA probably prev e n t e d t h e r e s o l u t i o n of m i t o c h o n d r i a l f r o m c h l o r o p l a s t D N A i n p r o f i l e c (fig. 3). A d e n a t u r a t i o n -
DNA.
Euglena
1017
mately the same buffer molarity, and therefore the s a t e l l i t e D N A f r o m t h e w i l d - t y p e is a m i x t u r e of chloroplast and mitochondrial DNA. O r g a n e l l e
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Fro. 5. - - Seannings of pooled DNA f r a c t i o n s f r o m the h y d r o x y a p a t i t e e h r o m a t o g r a m of figure 4. a) 31-33 ; b) 34-38 ; c) 39-41 ; d) 42-5,0. Centrifugalion conditions in n e u t r a l CsC1 are given in figure 2. r e n a t u r a t i o n e x p e r i m e n t w i t h s a m p l e c (fig. 3) a n d s a m p l e d (fig. 5) c o n f i r m s t h i s a s s u m p t i o n ; t h e respe~,(ive p r o f i l e s a r e d i s p l a y e d i n f i g u r e 6. T h e D N A f r a c t i o n f r o m t h e w i l d - t y p e d e n a t u r e s as o n e b r o a d p e a k ( p r o f i l e c) (~ = 1.70,0 g / c m 3) w i t h a s h o u l d e r at 1.720 g / c m 3. A f t e r r e n a t u r a t i o n , h o w e v e r , a s h a r p m a j o r p e a k (1.6~87 g / c m a) a n d a n e v i d e n t s h o u l d e r (1.696 g / c m 3) a p p e a r s , i n a d d i t i o n to t h e m i n o r n u c l e a r D N A p e a k (1.717 g / c m 3 ) p r o f i l e c'). T h e s a t e l l i t e f r o m t h e b l e a c h e d m u t a n t s h o w s , a f t e r d e n a t u r a t i o n ( p r o f i l e d), a s h o u l d e r of d e n s i t y 9 = 1.700 g / c m ~ a n d a m a j o r n u c l e a r D N A p e a k (9 = 1.72.0 g / c m 3 ) ; a f t e r r e n a t u r a t i o n ( p r o file d ' ) a s h a r p p e a k (9 ---- 1.696 g / c m a ) r e p l a c e s t h e b r o a d s h o u l d e r of p r o f i l e d a n d a m a j o r n u c l e a r D N A p e a k (~ : 1.717 g / c m 3 ) . C o m p a r i n g p r o f i l e c' w i t h d ' o n e r e a d i l y sees t h a t t h e s h o u l d e r in the former matches the renatured satellite (mit o c h o n d r i a l DNA) of t h e b l e a c h e d m u t a n t . T h e c o n c l u s i o n is t h a t b o t h c h l o r o p l a s t a n d m i t o c h o n driaI DNA elute from hydroxyapatitc at approxi-
BIOCHIMIE, 1972,
54, n ° 8.
I
1.700 1.720
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1.742
I 1.742
FIG. 6. - - Scannings of h e a t - d e n a t u r e d and r e n a t u r e d DNA from f r a c t i o n (c) of t h e b a t c h e x p e r i m e n t (see figure 3) a n d f r a c t i o n (d) f r o m e h r o m a t o g r a m of t h e bleached m u t a n t DNA (see figure 4). c and c' are the d e n a t u r e d a n d r e n a t u r e d DNA, respective.ly, from. f r a c t i o n (c) ; d a n d d' are the d e n a t u r e d a n d r e n a t u r e d D.NA from fraction (d), respectively. C e n t r i f u g a t i o n conditions in n e u t r a l CsC1 as given in figure 2.
DNA's should be prepared therefore from bleached mutants for mitochondrial DNA and from chlorop l a s t e n r i c h e d f r a c t i o n s f o r c h l o r o p l a s t DNA. In subsequent work, we investigated the chrom a t o g r a p h i c p u r i f i c a t i o n of c h l o r o p l a s t - e n r i c h e d f r a c t i o n s . I n t h e c o u r s e of t h e s e e x p e r i m e n t s s o m e
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Fro. 7. - - C h r o m a t o g r a p h y of DNA from a chlorop l a s t - e n r i c h e d f r a c t i o n of Euglena gracilis. 7 ml of DNA solution in 0.01M sodium p h o s p h a t e buffer (5.7 A_~o units) were loaded on a 1.08 × 60' em h y d r o x y a p a t i t e column e q u i l i b r a t e d w i t h the same buffer. The column was w a s h e d w i t h 30 ml of this buffer and 0.01 to 0.5 M g r a d i e n t (100'0 ml) was applied. 4.0 ml f r a c t i o n s were collected. Flow rate was 28 m l / hr. A~o recovery was 82 p. cent. Circles indicate the l i m i t s of the fractions w h i c h were pooled together.
E. S t u t z a n d G. B e r n a r d i .
1018
n o v e l i n f o r m a t i o n w a s o b t a i n e d on t h e s a t e l l i t e D N A w h i c h w a s r e c e n t l y d e s c r i b e d to o c c u r in c h l o r o p l a s t - e n r i c h e d f r a c t i o n s f r o m Euglena [17]. Two different chloroplast DNA preparations w e r e used, t h e first o n e still h e a v i l y c o n t a m i n a t e d
d
I I I I I I 1 I bl I
b y n u c l e a r DfNA, t h e s e c o n d o n e w i t h no signific a n t a m o u n t of n u c l e a r DNA (p = 1.707 g / c m a ) . Figure 7 shows the chromatogram obtained with t h e first p r e p a r a t i o n ; t h e e l u t i o n p r o f i l e s h o w s t w o d i s t i n c t p e a k s w h i c h c o r r e s p o n d to n u c l e a r D N A an d c h l o r o p l a s t DNA, r e s p e c t i v e l y . I n fact, this profile almost mimics the buoyant density p r o f i l e of t h e s t a r t i n g m a t e r i a l (fig. 8, a), c o n f i r m i n g t h e r e b y t h a t t h e c o l u m n is q u i t e e f f i c i e n t in s e p a r a t i n g t h e t w o t y p e s of D,NA. T h i s is confirm e d b y t h e b u o y a n t d e n s i t y a n a l y s i s of t h e five f r a c t i o n s b, c, d, e, f, w h i c h is in e s s e n c e g a v e a
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1.685! I 1.705 I I.~2 Fro. 8. - - Seanning s of pooled DNA fractions from the chromatogram of figure 7. The scanning of the starting ma'terial is shown in profile (a). b) 100-10.6 ; c) 107-111) ; d) 111-114 ; e) 115-117 ; f) 118-127. Centrifugation conditions in neutral CsC1 density gradients as given in figure 2. 1.685
L707 1.742
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FRACTION NUMBER F1G. 10. - - Chromatography of DNA from chloroplast fraction of Euglena gracilis. 4.6 ml of DNA solution in 0.1 M sodium phos,phate buffer (3.6 A~o units) were loaded on a 1:08 X 60 cm hydroxyapatite column equilibra, ted 'with 'the same buffer (3,0 ml) and a 0.1 to 0.5 M gradient (800 ml) was applied. 5.5. ml fractions were co.llected. Flow rate was 33 m l / h r . A~o units recovery was 95 p. cent. Circles indicate the limits of the fractions which were pooled together.
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FIG. 9. Scannings of heat-denatured b, c, d, e and renatured b', e', d', e, pool.ed DNA fractions from the chromatogram of figure 7. CeI~trifugation conditions in neutral CsC1 as given in figure 2. Pooled fractions are indicated as in figure 8.
BIOCHIMIE, 19,72, 54, n ° 8.
s i m i l a r r e s u l t as t h e first c h r o m a t o g r a m w i t h a p r o p o r t i o n a t e up s h i f t of c h l o r o p l a s t DNA. Again, t h e e a r l y e l u t i n g f r a c t i o n (b, c) c o n t a i n o n l y the n u c l e a r DNA, t h e l a t e r f r a c t i o n s (e, f), c o n t a i n m a i n l y t h e l i g h t e r c h l o r o p l a s t DNA. T h e i n t e r m e d i a t e f r a c t i o n (d) is a m i x t u r e of h e a v y a n d l i g h t DNA. The DNA samples were characterized by their denaturation-renaturation behavior. The results a r e g i v e n i n figure 9. P a t t e r n s b, c, d, a n d e b e l o n g to t h e d e n a t u r e d D N A ; p a t t e r n s b', c', d', a n d e b e l o n g to t h e r e n a t u r e d DNA. All d e n a t u r e d D N A s a m p l e s s h o w t h e t y p i c a l i n c r e a s e in b u o y a n t d e n s i t y of a p p r o x i m a t e l y 15 rag. T h e s a m p l e s differ, h o w e v e r , in t h e i r r e n a t u r a t i o n b e h a v i o r s . As exp e c t e d [28], t h e c h l o r o p l a s t D N A (e, e') r e g a i n s
Hydroxyapatite c h r o m a t o g r a p h y of E u g l e n a DNA. almost c o m p l e t e l y the density of the native DNA (1.687 versus 1.185 g/cm3). The n u c l e a r DNA (b, b') s h o w e d after r e n a t u r a t i o n a b r o a d p e a k w i t h a m e a n b u o y a n t d e n s i t y of 1.718 g/cm3. The profile of the t w o i n t e r m e d i a t e f r a c t i o n s reveal t w o
I I I
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F i g u r e 10 s h o w s the c h r o m a t o g r a m o b t a i n e d w i t h the s e c o n d c h l o r o p l a s t DNA p r e p a r a t i o n . As a l r e a d y m e n t i o n e d , in this case (not shown) nuclear DNA (1.707 g/cm3) w a s not detectable, but the satellite DNA (1.700 g / c m 3) w a s v e r y p r o m i nent. F i g u r e 11 s h o w s the b u o y a n t d e n s i t y profiles o b t a i n e d on p o o l e d f r a c t i o n s f r o m the chrom a t o g r a m . It is e v i d e n t that f r a c t i o n s eluttng at i n c r e a s i n g l y h i g h e r m o l a r i t i e s b e c o m e e n r i c h e d in c h l o r o p l a s t DNA w i t h c o n c o m i t a n t d e c r e a s e of satellite DNA. I
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I
1.685 1.699
I
I I 1.742 Fro. 11. - - Scannings of pooled DNA fractions from the chromatogram of figure 10. a) 52-59 ; b) 60-61 : c) 6,2-65 ; d) 66-73. Centrifugation conditions in neutral CsCl density gradient as gi'ven in figure 2.
Fro. 12. - - Preparative CsC1 density gradient of (a) satellite DNA (2.28 A~o units) (b) ehloroplast DNA (0.94 A~o units). The DN,A preparations used in this experiment were obtained from the pooled a + b and c + d fractions of the chromatogram shown in figure 10, respectively. Divisions on the abseissa scales correspond to 1.93 ml. Arrows indicate the central fractions which were used for analytical gradient (fig. 13) and base analysis (table I). ¢1
c o m p o n e n t s after r e n a t u r a t i o n s . Profile c' s h o w s a s h a r p p e a k w i t h a d e n s i t y of 1.703 g / c m a and a b r o a d e r c o m p o n e n t s w i t h an a v e r a g e density of 1.714 g / c m 3. The sample d' contains p r o b a b l y some c h l o r o p l a s t DNA (1.688 g/cm3) and a comp o n e n t of density 1.700. g / c m 3, p r o b a b l y i d e n l i fiable w i t h satellite DNA r e c e n t l y d e s c r i b e d [17]. The results of figure 9 suggest that the satellite !)NA is eluted f r o m h y d r o x y a p a t i t e at an i n t e r m e diate p o s i t i o n b e t w e e n n u c l e a r and c h l o r o p l a s t DNA, a c o n c l u s i o n c o n f i r m e d by the f o l l o w i n g experiments. TABLE 1.
Nucleoside analysis o[ chloroplast and satellite DNA's. I G i__
T
C
/', A
G + C
- - i - -
I Chloroplast DNA(a)
12.912.9
37.8 37.4
12 1 !i 37.2 12.2 [ 37.5
25.0 25,1
Satellite DNA (b)
2122.76
28.6 28.9
19.2 19,2
41.8 40.9
29.5 30.2
(u) Duplicate analysis on Bio Gel-2 coluinn. (b) Duplicate analysis on Aminex A6 column. BIOCHIMIE, 19,72, 54, n ° 8.
I
1.684 I 1.742 1.700 FiG. 13. - - Analytical GsG1 density gradient of (a) satellite DNA (b) chloroplast DNA. The DNA preparations used in this experiment originated from the centra.1 fractions of the preparative gradients shown in the previous figure.
F r a c t i o n s a + b and c + d f r o m the c h r o m a t o g r a m of figure 10 w e r e f u r t h e r purified by p r e p a r a t i v e CsCl density g r a d i e n t s (fig. 12). F i g u r e 13 s h o w s the a n a l y t i c a l s c a n n i n g s of these p u r i f i e d fractions, c o r r e s p o n d i n g to satellite (~ = 1.700 g/ era3), a n d c h l o r o p l a s t (~ -- 1.785 g/cm3) DNA. N u c l e s i d e analysis for these p r o d u c t s are s h o w n in table I. T h e GC levels found f o r c h l o r o p l a s t and
E. S t u t z a n d G. Bernardi.
1020
s a t e l l i t e D N A w e r e 25 p. c e n t a n d 40 p. c e n t , r e s pectively, in good agreement with the buoyant d e n s i t y r e s u l t s . T h e s e d i m e n t a t i o n c o e f f i c i e n t s of t h e t w o D N A ' s w e r e 218S a n d 28 S, r e s p e c t i v e l y .
DISCUSSION.
Acknowledgments. The Euglena c h l o r o p l a s t a n d m i t o c h o n d r i a l DNA samples were p r e p a r e d b y Mr. J. P. V a n d r e y a n d Mr. E. J. Crouse, N o r t h w e s t e r n U n i v e r s i t y ; t h e technical assistance of Miss C. G a i l l a r d is graCefully acknowledged. This w o r k w a s supported, i n part, b y g r a n t GB 19191 from the N a t i o n a l Science F n u n d a t i o n a n d a special Research G r a n t f r o m N o r t h w e s t e r n U n i v e r s i t y to one of u s (E.S.).
The present work establishes chromatography o n h y d r o x y a p a t i t e as a n e w t o o l f o r t h e p r e p a r a t i o n of b o t h m i t o c h o n d r i a l a n d c h l o r o p l a s t D N A ' s f r o m Euglena gracilis. I n a d d i t i o n , a s a t e l l i t e DN,A h a v i n g a b u o y a n t d e n s i t y of 1.700 g / c m 3 c a n also be separated from chloroplast-enriched preparat i o n s . T h e c e l l u l a r o r i g i n of t h i s s a t e l l i t e D N A will be discussed elsewhere (paper in preparation).
Nous avons gtudi6 ]e c o m p o r t e m e n t e h r o m a t o g r a p h i q u e des DNAs n a t i f s d'Euglena gracilis sur eolonnes d ' h y d r o x y a p a t i t e . Des p r g p a r a t i o n s d'acides nuclgiques t o t a u x peuvent ~tre fractionn~es sur colonne, en DNA nucIdaire et DNA des o r g a n i t e s ( m i t o e h o n d r i e s e.t chloroplastes). Une s~paration en <> p e r m e t d'obten i r r a p i d e m e n t le DNA des o~ganites ~t p a r t i r de pr~para¢ions d?aeides uuel~iques; totaux.
T h e p r a c t i c a l a d v a n t a g e s of t h e h y d r o x y a p a t i t e chromatography, particularly in its batchwise oper a t i o n , h a v e b e e n d i s c u s s e d e l s e w h e r e El0] a n d are too obvious, especially for large-scale preparations, to be further commented upon here.
La c h r o m a t o g r a p h i c de p r 6 p a r a t i o n s de DNA de m u t a n t s 6tiol6s p a r la c h a l e u r p e r m e t de s6parer le DNA m i t o c h o n d r i a l du DNA nucl6aire. La c h r o m a t o graphic de p r g p a r a t i o n s de DNA o b t e n u e s h p a r t i r de f r a c t i o u s d'Eugl~ne enrichies en chlorolalastes p e r m e t de prdparer le D NA des cbioroplastes et, en outre, u n DNA satellite d~jh d~crit (p ---- 1.7,00, g/cm~).
I t m a y b e i n t e r e s t i n g to r e m a r k t h a t t h e f o u r D N A ' s f r o m Euglena i n v e s t i g a t e d h e r e , n a m e l y t h e n u c l e a r DNA, t h e s a t e l l i t e D N A (? = 1.70.0 g / e m a ) , mitoehondrial DNA and chloroplast DNA are evidently eluted in the order of increasing AT cont e n t s . S o m e s a t e l l i t e D N A ' s also a p p e a r to b e e l u t e d a c c o r d i n g to t h e i r A T c o n t e n t s : f o r i n s t a n c e , s a t e l lite I a n d I I f r o m c a l f t h y m u s , w h i c h a r e r i c h e r i n GC c o m p a r e d w i t h m a i n b a n d DNA, a r e e l u t e d at a l o w e r m o l a r i t y t h a n t h e l a t t e r [5-7] ; m o u s e s a t e l l i t e w h i c h is p o o r e r i n GC c o m p a r e d w i t h m a i n b a n d D N A is e l u t e d a f t e r t h e l a t t e r [6, 7]. I t m i g h t b e t e m p t i n g to c o n c l u d e t h e r e f o r e t h a t A T - r i c h D N A ' s a r e e l u t e d f r o m h y d r o x y a p a t i t e at h i g h e r m o l a r i t i e s t h a n G C - r i e h D N A s, as t h e y a r e from methylated serum albumin - kieselguhr c o l u m n s [29], p a r t i c u l a r l y i n v i e w of t h e d i f f e r e n t secondary structure reported or suggested for very A T - r i c h D N A ' s [30, 31]. S u c h a c o n c l u s i o n i n all l i k e l i n e s s is n o t of g e n e r a l v a l i d i t y s i n c e t h e elut i o n m o l a r i t i e s of n a t i v e D N A ' s f r o m h y d r o x y a p a t i t e c o l u m n s a r e p r o b a b l y r e l a t e d t o s t r u c t u r a l features which do not depend upon the overall base c o m p o s i t i o n i n a s i m p l e w a y . I t is p o s s i b l e , h o w e ver, that the high elution molarity from hydroxya p a t i t e is a p r o p e r t y c o m m o n to m a n y A T - r i c h D N A ' s a n d r e f l e c t s t h e p r e s e n c e i n t h e s e D~NA's of short alternating and non-alternating AT sequences ; i n v e s t i g a t i o n s of o t h e r A T - r i c h D N A ' s s u c h as t h e m i t o c h o n d r i a l D N A ' s f r o m Tetrahgmena pgriformis [32] o r Physarum polgcephalum [33] might be interesting in this connection.
BIOCHIM1E, 19.72, 54, n ° 8.
Rgsu~.
Nous pr6seutons ici quelques propri4t~s p h y s i q u e s et c h i m i q u e s des; DNAs d'Eugl6ne o b t e n u s p a r c h r o m a tographic sur h y d r o x y a p a t i t e . ZUSAMMENFASSUNG. W i r h a b e n das B e n e h m e n der n a t i v e n DNA yon
Euglena gracilis an H y d r o x y a p a t i t s / i u l e n nnters.ucht. Pr~iparate von g e s a m t e n N,ukleins~iuren k 6 n n e n a n einer S~iule in Nukleus- u n d Organellen (Mitoehondrien u n d Chloroplasten)~-DNA f r a k t i o n i e r t werden. Eine <>-Trennung e r l a u b t es die Organellen-DNA aus Pr~iparaten yon g e s a m t e n Nukleinsfiuren rasch zu erhalten. Die C h r o m a t o g r a p h i c yon P r i i p a r a t e n der DNA a~s d u r c h Hitze v e r b l a s s t e n M u t a n t e n e r l a u b t die Trenn u n g der Mitochondrien-DNA yon der Nukleus-DNA. Die C h r o m a t o g r a p h i c tier aus. E u g l e n f r a k i i o n e n , welche an Chlorop~asten a n g e r e i e h e r t Wurden, e r h a l t e n e n DNA-Pr~iparate e r l a u h t es die Chloroplasten-DNA zu e r h a l t e n u n d a u s s e r d e m eine schon b e s c h r i e b e n e Satellit-DNA (9 : 1 700 g/cma). W i r stellen h i e r einige p h y s i k a l i s e h e n u u d chemist h e E i g e n s c h a f t e n der Euglen-DNA vor, w.elche d u r c h Chromatographic an Hydroxyapatit erhalten wurden.
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Hydroxyapatite
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