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Separation and characterization of Euglena gracilis chloroplast single-strand DNA
16
BIOCHIMICAET BIOPHYSICAACTA
BBA 96488
S E P A R A T I O N AND C H A R A C T E R I Z A T I O N OF E U G L E N A
GRACILIS
C H L O R O P L A S T S I N G L E - S T R A N D DNA
ERHARD STUTZ AND JAMES R. RAWSON* Department of Biological Sciences, Northwestern University, Evanston, Ill. 6020± (U.S.A.) (Received December ist, 1969)
SUMMARY Euglena gracilis chloroplast DNA when equilibrated in an alkaline CsC1 densit y gradient yields two clearly separated bands differing in alkaline buoyant density by 0.023 g/cm 3. These bands are separated preparatively and shown to represent single-strand DNA's differing in neutral buoyant density and renaturing characteristics. Both heavy and light single-strand DNA fractions are hybridized with a2p_ labeled chloroplast ribosomal RNA (filter). The heavy fraction retains approx. IO times more ribosomal RNA than the light DNA fraction. This indicates that tile majority of base sequences coding for chloroplast ribosomal RNA is contained ill the heavier of the two single-strand DNA fractions.
INTRODUCTION It is firmly established 1-3 that chloroplasts of Euglena gracilis contain a double-strand DNA quite different in buoyant density from the nuclear DNA, thereby permitting it to be easily separated from the nuclear DNA in a preparative CsC1 density gradient. Purified chloroplast DNA shows in an analytical neutral CsC1 density gradient a single sharp band with a buoyant density of 1.685 g/cm 3 (refs.I-3). We found, however, the same DNA when analyzed in an alkaline CsCI gradient to reproducibly yield two broad but clearly separated bands. This suggests that Euglena chloroplast DNA, similar, for example, to certain mitochondrial DNA ~ or nuclear satellite DNA 5,n might be split into two classes of DNA molecules of different densities and complenlentary to each other. It has been shown that in certain genomes only the heavy DNA strand or strand segments contain the coding base sequences 7,8. Thus, the question arises as to whether there is any functional difference between the two Euglena chloroplast DNA's banding with different densities. To answer this question we isolated in preparative amounts a high and a low density single-strand DNA fraction and hybridized each with Euglena chloroplast ribosomal RNA. We show that the heavy DNA fraction contains the majority of strand segments coding for chloroplast ribosomal RNA. * Present address: University of Chicago, Department of Biophysics, Chicago, [11., U.S.A. Biochim. Biophys. ,qcta, 209 (197o) 16-23
EUGLENA CHLOROPLAST SINGLE-STRAND D N A
17
MATERIALS AND METHODS
Cell growth Euglena gracilis Klebs (Z strain) cells were routinely grown under autotrophic conditions, harvested, washed and stored at --6o ° as reported earlier °. These cells were used for DNA and RNA isolation.
DNA isolation Chloroplasts were isolated as described earlied °. To a chloroplast pellet obtained from approx. 35 g packed cells 14 ml of buffer containing IOO mM Tris-HC1 (pH 7.5), 2.5 % sodium dodecyl sulfate, and IO mM E D T A were added and the suspension heated for IO min at 60 °. After cooling, 4 M sodium perchlorate was added to a final concentration of I M. 2 vol. of a chloroform-isoamylalcohol (24:1, v/v) 11 mixture were added and the emulsion was shaken for I h at room temperature. The aqueous and organic phases were separated b y centrifugation. DNA fibers were spooled from the chilled aqueous phase after addition of 2 vol. of ethanol. The DNA was resuspended in 15 mM NaCI-I. 5 mM sodium citrate and incubated with IOO/~g ribonuclease/ml (ribonuclease B, Bovine Pancreas, Type VII, Sigma) at 37 °, I h. The solution was then deproteinized b y shaking successively with phenol and chloroform-isoamylalcohol for IO rain in each case. The DNA fibers were finally spooled from the aqueous phase after addition of 2 vol. of cold ethanol. Such a preparation gave approx. 1-2 mg of DNA consisting of one part of nuclear DNA (p = 1.7o8 g/cm 3, neutral CsC1) and one part of chloroplast DNA (p = 1.685 g/cm 3, neutral CsC1). Several such batches were combined and the chloroplast DNA was separated from the nuclear components in a preparative neutral CsC1 density gradient according to FLAMM et al. 1~. The two peak fractions were separately pooled, dialyzed overnight against 15 mM NaCI-I. 5 mM sodium citrate and o.I inM EDTA. The volume of the dialyzate was brought down b y flash evaporation, and the pure chloroplast and nuclear DNA spooled in the usual way from cold aqueous solutions (approx. 0.5 mg DNA/ml) after addition of cold ethanol. The DNA fibers were redissolved in 15 mM NaCI-I. 5 mM sodium citrate and stored at --18 ° (0.5 mg/ml). The purity of the DNA was checked in analytical CsC1 density gradients.
Separation o/ single strand DNA Purified chloroplast DNA was denatured with alkali and separated in a preparative alkaline CsC1 density gradient according to FLAMM el al. 13. Purified chloroplast DNA (12o-15o #g) was denatured at room temperature in 3.6 ml of 15 mM NaCI-I.5 mM sodium citrate which had been adjusted to p H 12 with NaOH. o.I °/o sodium dodecyl sulfate was added (0.5 ml) plus solid CsC1 to give a final solution (4.5 ml) with a starting density of 1.71o g/cm 3. The samples were centrifuged in Polyallomer tubes in a Spineo 40 rotor for 72 h, 25 °, 33 ooo rev./min. The equilibrated gradients were monitored b y piercing the tubes with a 25-gauge needle and collecting fractions of approx. 0.04 ml (two drops). The dropping rate was controlled b y a H a r v a r d precision p u m p adjusted to a flow rate of 0.58 ml/min. 0.5 ml of 15 mM NaCI-I.5 mM sodium citrate was added to each fraction and the absorbance at 260 m# measured. The peak fractions were collected, dialyzed against 15 mM NaC11.5 mM sodium citrate and o.I mM E D T A in the cold and finally evaporated to a B i o c h i m . B i o p h y s . Acta, 209 (197 ° ) 16-23
18
E. STUTZ, j. R. RAWSON
small volume in vacuo. These solutions containing between 4 ° 5o/zg of DNA/ml were stored at --18 ° and used without further purification. Labeling and isolation o/chloroplast r R N A The isolation and purification of Euglena chloroplast ribosomal RNA has been described in detail 1°. Cells were grown for 8 days in Difco Euglena Broth in the dark at 25-27 ° up to 2" lO 6 cells/ml. The cells were transferred into 5-gallon carboys containing 6.5 1 of autotrophic medium, according to CRAMER AND MEYERS 14, reduced in inorganic phosphate 50 times. Carrier-free 32P1 (New England Nuclear) was added to a final concentration of 3.1 mC/1. The medium was initially saturated with 5 }~ CO2-air and the cells were grown in constant light (15o ft candles) at 25-27 ° for 48 h. The ribosomal RNA was isolated from purified ribosomes and separated from low-molecular-weight RNA and transfer RNA on a sucrose gradient. The specific radioactivity of the final ribosomal RNA was in tile range of 3000-5o00 counts/min per I/~g RNA. Itybridization The technique was basically that of GILLESPIE AND SPIEGELMAN15. Alkalinedenatured or single-strand fractionated DNA was dissolved in 0.30 M NaCl-o.o3o M sodium citrate (approx. 5/~g/ml) and 5 ml passed through a nitrocellulose filter (S and S B6, 25 mm). The DNA retained on the filter was calculated from the difference in absorbance of the starting solution and the eluate. Possible losses of DNA during incubation were not measured. Charged and blank filters were incubated together in screw cap plastic vials. After incubation all filters were first rinsed 3 times in 5 ° m l of 0.30 mM NaCl-o.o3o mM sodium citrate, then incubated for I h at room temperature in 5 ml 0.3o mM NaCl-o.o3o mM sodium citrate containing 20 #g ribonuclease/ml, washed again in excess of 0.30 M NaCl-o.o3o M sodium citrate and after drying monitored for radioactivity in a Tri-Carb counter, Model 3003, using scintillating fluid (15 ml per sample) containing IO parts of toluene scintillation liquid (4 g 2,5diphenyloxazole (PPO) and IOO mg 1,4-bis-(5-phenyloxazolyl-2)benzene (POPOP) per I toluene) and 6 parts ethylene glycol monoethyl ether.
RESULTS
Purified Euglena chloroplast DNA gave in the analytical neutral CsC1 density gradient a single sharp peak with a buoyant density of 1.685 g/cm 3 (Fig. Ia) which is consistent with earlier reports 1-~. When the same DNA was alkaline denatured and rapidly neutralized in the cold, its density increased to 1.7o 3 g/cm 3 and the band broadened considerably (Fig. Ib). The buoyant density of such denatured DNA when incubated for I h at 63-64 ° in 0.30 M NaCl-o.o3o M sodium citrate shifted back to 1.687 g/cm 3, indicating extensive reannealing (Fig. IC). However, when chloroplast DNA was analyzed in an alkaline CsC1 density gradient two bands appeared with buoyant densities of 1.729 g/cm 3 and 1.752 g/cm ~ as calculated from the limiting isoconcentration distance and the initial density of the solution 16 (Fig. Id). Several chloroplast DNA preparations were tested in this way, and they all consistently showed the same double banding pattern. Biochim. Biophys. Acta,r2o9 (197o) 16-23
EUGLENA CHLOROPLAST SINGLE-STRAND DNA
19
a
0.16
v 1.687
1.703
z o.os
1.742
1.885 i
i
1.729
1.752
i
t
FRACTION
t
J
NUMBER
Fig. i. Density equilibrium sedimentation of Euglena chloroplast DNA in CsC1. a, 2/~g native DNA; b, 5/2g DNA denatured in o.13 ml 15 mM NaCI-I. 5 mlV[ sodium citrate adjusted to pH 12, io min, room temperature, chilled in ice and rapidly neutralized; c, 3/~g DNA renatured in o.13 ml, o.3o M NaCl-o.o3o M sodium citrate, p H 7, i h, 63°; d, 5 #g DNA denatured in o.13 ml 15 mM NaCI-I. 5 mM sodium citrate adjusted to pH 12, IO rain, room temperature. Centrifugation: Spinco Model E, An-D rotor, 44 77 ° rev./min, 18-2o h, 20°; tracings a, b, c are aligned with respect to the reference DNA peak, [15NJDNA of Pseudomonas aeruginosa (density z.742 g/cma). The densities in the neutral CsC1 gradients (a, b, c) are calculated relative to the reference according to SUEOKA19. The refractive indices of the neutral CsC] solution were kept in the range of 1.4ooo ±0.oo2. The densities of the samples in the alkaline gradient, d, were calculated as mentioned in the text. Fig. 2. Separation of single strands of Euglena chloroplast DNA. lO5/~g of Euglena chloroplast DNA were alkaline denatured, equilibrated in an alkaline CsC1 density gradient and fractionated as described in the text. The heavy and light fractions were pooled as indicated by the arrows. In the average of several such gradients the ratio of h e a v y to light single-strand DNA fraction was 4 to 3 and the overall recovery of DNA between 42 and 48 %. We isolated high and low density single-stranded chloroplast DNA in preparat i v e a m o u n t s u s i n g t h e m e t h o d of FLAMM el al. 13. Fig. 2 s h o w s t h e e l u t i o n p r o f i l e of s u c h a g r a d i e n t . Tile b a n d s a r e b r o a d a n d i n d i c a t e a d e n s i t y - p o p u l a t i o n of s t r a n d s o r s t r a n d f r a g m e n t s r a t h e r t h a n h o m o g e n o u s f r a c t i o n s of i n t a c t h e a v y a n d l i g h t s t r a n d s . T h e a r r o w s m a r k t h e r a n g e of t u b e s p o o l e d . I d e n t i c a l p e a k s f r o m s e v e r a l s u c h g r a d i e n t s w e r e c o m b i n e d a n d p r o c e s s e d a s m e n t i o n e d i n MATERIALS AND METHODS. Heavy and light fractions were tested in the analytical ultracentrifuge as shown i n Fig. 3- I n a s e r i e s of d e n a t u r i n g , r e n a t u r i n g a n d c o - r e n a t u r i n g e x p e r i m e n t s w e
Biochim. Biophys. Acta, 2o9 (197 ° ) 16-23
20
E. STUTZ, J. R. RAWSON
I
I
c
I
i
.
A
1.[
.
.
.
.
/)
oz
~o.~
L1.693 1.687
1.742
eiRNA
Fig. 3. Density equilibrium sedimentation of fractionated Euglena chloroplast DNA. a, 6/~g heavy fraction DNA, incubated in o.II ml 15 mlV[ NaCI-I.5 mM sodium citrate adjusted to pH 12, IO min, room temperature, chilled in ice, rapidly neutralized; b, 6/~g heavy fraction DNA incubated in o. 15 ml o.3o M NaCl-o.o3o M sodiumcitrate, pH 7, 6 h, 63°; c, 6/*g light fraction DNA, treated as mentioned sub a; d, 6 ktg light fraction DNA, treated as mentioned sub b; e, 3/~g heavy fraction plus 3/~g light fraction DNA in o.13 ml o.30 M NaCl-o.o3o M sodium citrate, pH 7, jointly incubated for 6 h, 63 °. Centrifugation, DNA reference and density calculation as mentioned in legend to Fig. i. Fig. 4. Saturation of chloroplast DNA filters with chloroplast ribosomal RNA. Filters loaded with 2o-2i/~g chloroplast DNA were challenged with increasing amounts of ribosomal RNA and incubated for 2o h at 64-65 `~in o.3o M NaCl-o.o3o ~ sodium citrate. t r i e d to e s t a b l i s h w h e t h e r a n d t o w h a t e x t e n t t h e h e a v y a n d light f r a c t i o n s c o n t a i n e d c o m p l e m e n t a r y s t r a n d s e g m e n t s . E a c h of t h e f r a c t i o n s w e r e d e n a t u r e d w i t h alkali, chilled a n d r a p i d l y n e u t r a l i z e d . W h e n a n a l y z e d in a n e u t r a l CsC1 d e n s i t y g r a d i e n t b o t h p r e p a r a t i o n s g a v e a r a t h e r b r o a d b a n d w i t h m e a n d e n s i t i e s of 1.7o 5 g / c m 3 (Fig. 3a) a n d 1.698 g / c m a (Fig. 3c), r e s p e c t i v e l y , t h e r e b y b r a c k e t i n g t h e n e u t r a l b u o y a n t d e n s i t y of d e n a t u r e d c h l o r o p l a s t D N A (Fig. I b ) . A l i q u o t s of t h e s a m e p r e p a r a t i o n s w e r e i n c u b a t e d for 6 h at 63 64 ° in 0.30 M N a C l - o . o 3 o M s o d i u m c i t r a t e a n d a g a i n a n a l y z e d in a n e u t r a l CsC1 g r a d i e n t . U n d e r s u c h r e n a t u r i n g c o n d i t i o n s t h e h e a v y f r a c t i o n s l i g h t l y c h a n g e d its o v e r a l l d e n s i t y as r e f l e c t e d in t h e r e s p e c t i v e t r a c i n g (Fig. 3b), s h o w i n g a s h o u l d e r on t h e left side. Also t h e l i g h t f r a c t i o n d e c r e a s e d its d e n s i t y s l i g h t l y u n d e r r e n a t u r i n g c o n d i t i o n s to a m e a n d e n s i t y of 1.693 g / c m 3 (Fig. Biochim. ldiophys. ,4cta, 209 (197 o) 16 23
E U G L E N A CHLOROPLAST SINGLE-STRAND
DNA
21
3d). But in both cases the bands remained broad, indicating that the majority of strand segments did not interact with each other. However, when equimolar amounts of the heavy and light fractions were combined and incubated under identical conditions, sharply banding DNA resulted with a density of 1.687 g/cm 3 (Fig. 3e). This value is close to the neutral buoyant density of native chloroplast DNA and identical with the value of renatured chloroplast DNA (see Fig. I). These data indicate that the heavy and light fractions represent populations of single-strand DNA which are essentially complementary to each other. In view of these results it became interesting to check whether there was any difference in the coding function between the two single-strand DNA fractions. We prepared highly purified Euglena chloroplast ribosomal RNA 1° which presumably is a product of the chloroplast genome. We hybridized it with total chloroplast DNA, to establish first the saturation value under a given set of incubation conditions (Fig. 4). Saturation is attained with 6-8/zg ribosomal RNA (22 S/I 7 S) per 2 o # g chloroplast DNA. The excess ribosomal RNA used to saturate 0.2 #g of DNA sites was 3o-4o-fold and below the 6o-fold excess needed under similar conditions in a bacterial system 15. Identical incubation conditions were used for hybridizing ribosomal RNA to fractionated chloroplast DNA (Table I). We saw that the heavy fraction
TABLE I H Y B R I D I Z A T I O N BETV*rEEN t?~UGLENA RIBOSOMAL
R N A AND VARIOUS T Y P E S OF E U G L E N A D N A
Specific a c t i v i t y , c o r r e c t e d for q u e n c h i n g : 145 c o u n t s / m i n pe r o . I / ~ g 22-S/I7-S r i b o s o m a l R N A
DNA source
Chloroplast heavy fraction
Chloroplast light fraction
DNA ~ug)
s2P-labeled ribosomal RNA source
RNA ~ug)
8 IO-I I
9-1o
Chloroplast 22 S/I 7 S Chloroplast 22 S/I 7 S
16 8 16
Chloroplast total
22
Chloroplast 22 S/I 7 S
8 16
Counts/rain per Jilter
Hybridization (%)
Charged
Blank
347 302 335 312
14 17 17 19
2.3 1.9 2.2 2.0
39 45 45 38
14 16 18 20
o.i 0.2 0.2 o.i
4o2 364 4o7 4o 7
25 21 19 21
1.2 i.o 1.2 1.2
retained approx. IO times more 32P-labeled ribosomal RNA than the light fraction (values corrected for blanks) and the percentage hybridization of the h e a v y fraction DNA is about twice the percentage value of total DNA. This indicates that the heavy DNA fraction contains the majority of base sequences coding for the 22-S/I7-S chloroplast ribosomal RNA. Biochim. B i o p h y s . ~tcta, 2o9 (197 ° ) 16-23
22
E. STUTZ, J. R. RAWSON
DISCUSSION
According to these results it is possible to split the Euglena chloroplast DNA in an alkaline CsC1 density gradient into two populations of single-strand DNA which differ in their overall alkaline buoyant density by o.o23 g/cm s. VINOGRAD eb al. 17 have shown that the buoyant density of denatured single-strand DNA sharply increases in the range of p H 12 as a function of the T + G content. They therefore suggested this procedure to discriminate between complementary strands of unequal base composition. The method proved feasible in case of relatively small and homogenous genomes as e.g., phage DNA is or mouse satellite nuclear DNA 13 where individual heavy and light strands could be separated. The Euglena chloroplast genome has a molecular weight of approx. 6.2. lO9 (ref. I) and is easily fragmented during the isolation. We would, therefore, expect that each of the two single-strand DNA fractions we have isolated contain strand fragments from both in vivo chromosomal strands. Comparing the G + C content of chloroplast ribosomal RNA which is 52 % (ref. IO) with the G + C content of chloroplast DNA which is 25 %, one realizes that the ribosomal RNA cistrons are quite different from the overall DNA base composition. In fact, the chances for the ribosomal RNA cistrons ( G + T = 45 ~'o) to band in the heavier of the two alkaline single-strand DNA fractions ( G + T -- 57 °/o) are marginal unlessT- or G-rich sequences are contiguous to the cistrons. If such additional sequences are removed because of strand fragmentation the cistrons would tend to band in the light alkaline single-strand DNA fraction. Such m a y be the reason for the IO ~o transcribing base sequences found in the light fraction. Under saturation conditions 1.2 }"o of the chloroplast DNA hybridizes with 22-S/I7-S RNA. This is close to the I °/o reported by SCOTT AND SMILLIEg0. We know from competition experiments that the annealing reaction is specific. On this basis and taking the size of the Euglena DNA as 6.2.Io 9 (tool. wt.) the number of cistrons coding for 22-S/I7-S RNA is about twenty. Each of the two isolated single-strand DNA preparations displays a certain renaturing capacity as reflected by the reverse shift in neutral buoyant density. This m a y be due to incomplete separation of the complementary strand fragments and/or intra-strand annealing as was, e.g., shown for mouse single-strand satellite DNA 5. Nothing is known at present about regional differences in base composition or possible secondary structure formation in single-strand chloroplast DNA. The quality of the alkaline separated single-strand DNA fraction might, therefore, change with the fragment size of the DNA preparation. BORST AND AAIJ 7 have shown that rat liver mitochondrial DNA undergoes strand separation in an alkaline CsC1 density gradient and the heavy strand acts as exclusive messenger strand under the chosen experimental conditions. OISHIs has reported that Bacillus subtilis heavy strand DNA which was isolated by using the methylated albumin kieselguhr column 21 hybridizes with ribosomal and transfer RNA exclusively. He concludes combining these data with earlier results from KUBINSKY et al. ~2 that all species of B. subtilis RNA are complementary to the heavy strand segments. It remains to be shown whether the heavy fraction of Euglena chloroplast DNA also codes for all species of RNA.
Biochim. Biophy$. Acta, 209 (197 o) 16--23
EUGLENA CHLOROPLAST SINGLE-STRAND
DNA
23
ACKNOWLEDGMENTS
The Pseudomonas aeruginosa was a gift from Dr. N. Welker. We thank Mrs. K. Patel for skillful technical help. This investigation was supported by Biomedical Science Support Grant FR-o7o28 and a Predoctoral Fellowship I-FI-GI7-3774-o2 to J. R. R. from the National Institutes of Health. We are grateful to Dr. H. Null for additional support. REFERENCES I G. BRAWERMANN AND J. ~v[. EISENSTADT, Biochim. Biophys. Acta, 91 (1964) 477. 2 D. S. RAY AND P. C. HANAWALT, J. Mol. Biol., 9 (1964) 812. 3 M. EDELMAN, C. A. COWAN, H. T. EPSTEIN AND J. A. SCHIFF, Proc. Natl. Acad. Sci. U.S., 52 /I964) 1214. 4 G. CORNED, L. ZARDI AND E. POLLI, J. Mol. Biol., 36 (1968) 419 . .5 W. G. FLAMM, P. ])¢[. B. WKLKER AND M. McCALLUM, J. Mol. Biol., 4 ° (1969) 423 . 6 G. CORNED, E. GINELLI AND E. POLLI, J. Mol. Biol., 33 (1968) 331. 7 P. BURST AND C. AAIJ, Biochem. Biophys. Res. Commun., 34 (1969) 358. 8 M. OISHI, Proc. Natl. Acad. Sci. U.S., 62 (1969) 256. 9 J. R. RAWSON AND E. STUTZ, J. Mol. Biol., 33 (1968) 309. io J. R. RAWSON AND E. STUTZ, Biochim. Biophys. Acta, 19o (1969) 368. i i J. ~¢[ARI~IUR, J. Mol. Biol., 3 (1961) 208. 12 W. G. FLAMM, H. E. BOND AND H. E. BURR, Biochim. Biophys. Acta, 129 (1966) 31o. 13 W. G. FLAMM, IV[.•CCALLUM AND P. M. B. WALKER, Proc. Natl. Acad. Sci. U.S., 57 (1967) 1729 14 M. CRAMER AND J. MEYERS, Arch. Mikrobiol., 17 (1952) 384 . 15 D. GILLESPIE AND S. SPIEGELMAN, J. Mol. Biol., 12 (1965) 829. 16 J. VINOGRAD AND H. E. HEARST, Progr. Chem. Org. Nat. Prod., 20 (1962) 395. 17 J. VINOGRAD, J. MORRIS, N. DAVlDSON AND N. DOVE, Proc. Natl. Acad. Sci. U.S., 49 (1963) 12. 18 W. DOERFLER AND D. S. HOGNESS, J. Mol. Biol., 33 (1968) 635. 19 N. SUEOKA, J. Mol. Biol., 3 (1961) 31. 20 N. S. SCOTT AND R. N. SMILLIE, Biochem. Biophys. Res. Commun., 28 (1967) 598. 21 14. RunNER, J. D. KARKAS AND E. CHARGAFF, Proc. Natl. Acad. Sci. U.S., 60 (1968) 630. 22 H. I~UBINSKY, Z. OPARA-KUBINSKA AND W. SZYBALSKY, J. Mol. Biol., 20 (1966) 313 .
Biochim. Biophys. Acta, 2o9 (197 o) 16-23
BIOCHIMICAET BIOPHYSICAACTA
BBA 96488
S E P A R A T I O N AND C H A R A C T E R I Z A T I O N OF E U G L E N A
GRACILIS
C H L O R O P L A S T S I N G L E - S T R A N D DNA
ERHARD STUTZ AND JAMES R. RAWSON* Department of Biological Sciences, Northwestern University, Evanston, Ill. 6020± (U.S.A.) (Received December ist, 1969)
SUMMARY Euglena gracilis chloroplast DNA when equilibrated in an alkaline CsC1 densit y gradient yields two clearly separated bands differing in alkaline buoyant density by 0.023 g/cm 3. These bands are separated preparatively and shown to represent single-strand DNA's differing in neutral buoyant density and renaturing characteristics. Both heavy and light single-strand DNA fractions are hybridized with a2p_ labeled chloroplast ribosomal RNA (filter). The heavy fraction retains approx. IO times more ribosomal RNA than the light DNA fraction. This indicates that tile majority of base sequences coding for chloroplast ribosomal RNA is contained ill the heavier of the two single-strand DNA fractions.
INTRODUCTION It is firmly established 1-3 that chloroplasts of Euglena gracilis contain a double-strand DNA quite different in buoyant density from the nuclear DNA, thereby permitting it to be easily separated from the nuclear DNA in a preparative CsC1 density gradient. Purified chloroplast DNA shows in an analytical neutral CsC1 density gradient a single sharp band with a buoyant density of 1.685 g/cm 3 (refs.I-3). We found, however, the same DNA when analyzed in an alkaline CsCI gradient to reproducibly yield two broad but clearly separated bands. This suggests that Euglena chloroplast DNA, similar, for example, to certain mitochondrial DNA ~ or nuclear satellite DNA 5,n might be split into two classes of DNA molecules of different densities and complenlentary to each other. It has been shown that in certain genomes only the heavy DNA strand or strand segments contain the coding base sequences 7,8. Thus, the question arises as to whether there is any functional difference between the two Euglena chloroplast DNA's banding with different densities. To answer this question we isolated in preparative amounts a high and a low density single-strand DNA fraction and hybridized each with Euglena chloroplast ribosomal RNA. We show that the heavy DNA fraction contains the majority of strand segments coding for chloroplast ribosomal RNA. * Present address: University of Chicago, Department of Biophysics, Chicago, [11., U.S.A. Biochim. Biophys. ,qcta, 209 (197o) 16-23
EUGLENA CHLOROPLAST SINGLE-STRAND D N A
17
MATERIALS AND METHODS
Cell growth Euglena gracilis Klebs (Z strain) cells were routinely grown under autotrophic conditions, harvested, washed and stored at --6o ° as reported earlier °. These cells were used for DNA and RNA isolation.
DNA isolation Chloroplasts were isolated as described earlied °. To a chloroplast pellet obtained from approx. 35 g packed cells 14 ml of buffer containing IOO mM Tris-HC1 (pH 7.5), 2.5 % sodium dodecyl sulfate, and IO mM E D T A were added and the suspension heated for IO min at 60 °. After cooling, 4 M sodium perchlorate was added to a final concentration of I M. 2 vol. of a chloroform-isoamylalcohol (24:1, v/v) 11 mixture were added and the emulsion was shaken for I h at room temperature. The aqueous and organic phases were separated b y centrifugation. DNA fibers were spooled from the chilled aqueous phase after addition of 2 vol. of ethanol. The DNA was resuspended in 15 mM NaCI-I. 5 mM sodium citrate and incubated with IOO/~g ribonuclease/ml (ribonuclease B, Bovine Pancreas, Type VII, Sigma) at 37 °, I h. The solution was then deproteinized b y shaking successively with phenol and chloroform-isoamylalcohol for IO rain in each case. The DNA fibers were finally spooled from the aqueous phase after addition of 2 vol. of cold ethanol. Such a preparation gave approx. 1-2 mg of DNA consisting of one part of nuclear DNA (p = 1.7o8 g/cm 3, neutral CsC1) and one part of chloroplast DNA (p = 1.685 g/cm 3, neutral CsC1). Several such batches were combined and the chloroplast DNA was separated from the nuclear components in a preparative neutral CsC1 density gradient according to FLAMM et al. 1~. The two peak fractions were separately pooled, dialyzed overnight against 15 mM NaCI-I. 5 mM sodium citrate and o.I inM EDTA. The volume of the dialyzate was brought down b y flash evaporation, and the pure chloroplast and nuclear DNA spooled in the usual way from cold aqueous solutions (approx. 0.5 mg DNA/ml) after addition of cold ethanol. The DNA fibers were redissolved in 15 mM NaCI-I. 5 mM sodium citrate and stored at --18 ° (0.5 mg/ml). The purity of the DNA was checked in analytical CsC1 density gradients.
Separation o/ single strand DNA Purified chloroplast DNA was denatured with alkali and separated in a preparative alkaline CsC1 density gradient according to FLAMM el al. 13. Purified chloroplast DNA (12o-15o #g) was denatured at room temperature in 3.6 ml of 15 mM NaCI-I.5 mM sodium citrate which had been adjusted to p H 12 with NaOH. o.I °/o sodium dodecyl sulfate was added (0.5 ml) plus solid CsC1 to give a final solution (4.5 ml) with a starting density of 1.71o g/cm 3. The samples were centrifuged in Polyallomer tubes in a Spineo 40 rotor for 72 h, 25 °, 33 ooo rev./min. The equilibrated gradients were monitored b y piercing the tubes with a 25-gauge needle and collecting fractions of approx. 0.04 ml (two drops). The dropping rate was controlled b y a H a r v a r d precision p u m p adjusted to a flow rate of 0.58 ml/min. 0.5 ml of 15 mM NaCI-I.5 mM sodium citrate was added to each fraction and the absorbance at 260 m# measured. The peak fractions were collected, dialyzed against 15 mM NaC11.5 mM sodium citrate and o.I mM E D T A in the cold and finally evaporated to a B i o c h i m . B i o p h y s . Acta, 209 (197 ° ) 16-23
18
E. STUTZ, j. R. RAWSON
small volume in vacuo. These solutions containing between 4 ° 5o/zg of DNA/ml were stored at --18 ° and used without further purification. Labeling and isolation o/chloroplast r R N A The isolation and purification of Euglena chloroplast ribosomal RNA has been described in detail 1°. Cells were grown for 8 days in Difco Euglena Broth in the dark at 25-27 ° up to 2" lO 6 cells/ml. The cells were transferred into 5-gallon carboys containing 6.5 1 of autotrophic medium, according to CRAMER AND MEYERS 14, reduced in inorganic phosphate 50 times. Carrier-free 32P1 (New England Nuclear) was added to a final concentration of 3.1 mC/1. The medium was initially saturated with 5 }~ CO2-air and the cells were grown in constant light (15o ft candles) at 25-27 ° for 48 h. The ribosomal RNA was isolated from purified ribosomes and separated from low-molecular-weight RNA and transfer RNA on a sucrose gradient. The specific radioactivity of the final ribosomal RNA was in tile range of 3000-5o00 counts/min per I/~g RNA. Itybridization The technique was basically that of GILLESPIE AND SPIEGELMAN15. Alkalinedenatured or single-strand fractionated DNA was dissolved in 0.30 M NaCl-o.o3o M sodium citrate (approx. 5/~g/ml) and 5 ml passed through a nitrocellulose filter (S and S B6, 25 mm). The DNA retained on the filter was calculated from the difference in absorbance of the starting solution and the eluate. Possible losses of DNA during incubation were not measured. Charged and blank filters were incubated together in screw cap plastic vials. After incubation all filters were first rinsed 3 times in 5 ° m l of 0.30 mM NaCl-o.o3o mM sodium citrate, then incubated for I h at room temperature in 5 ml 0.3o mM NaCl-o.o3o mM sodium citrate containing 20 #g ribonuclease/ml, washed again in excess of 0.30 M NaCl-o.o3o M sodium citrate and after drying monitored for radioactivity in a Tri-Carb counter, Model 3003, using scintillating fluid (15 ml per sample) containing IO parts of toluene scintillation liquid (4 g 2,5diphenyloxazole (PPO) and IOO mg 1,4-bis-(5-phenyloxazolyl-2)benzene (POPOP) per I toluene) and 6 parts ethylene glycol monoethyl ether.
RESULTS
Purified Euglena chloroplast DNA gave in the analytical neutral CsC1 density gradient a single sharp peak with a buoyant density of 1.685 g/cm 3 (Fig. Ia) which is consistent with earlier reports 1-~. When the same DNA was alkaline denatured and rapidly neutralized in the cold, its density increased to 1.7o 3 g/cm 3 and the band broadened considerably (Fig. Ib). The buoyant density of such denatured DNA when incubated for I h at 63-64 ° in 0.30 M NaCl-o.o3o M sodium citrate shifted back to 1.687 g/cm 3, indicating extensive reannealing (Fig. IC). However, when chloroplast DNA was analyzed in an alkaline CsC1 density gradient two bands appeared with buoyant densities of 1.729 g/cm 3 and 1.752 g/cm ~ as calculated from the limiting isoconcentration distance and the initial density of the solution 16 (Fig. Id). Several chloroplast DNA preparations were tested in this way, and they all consistently showed the same double banding pattern. Biochim. Biophys. Acta,r2o9 (197o) 16-23
EUGLENA CHLOROPLAST SINGLE-STRAND DNA
19
a
0.16
v 1.687
1.703
z o.os
1.742
1.885 i
i
1.729
1.752
i
t
FRACTION
t
J
NUMBER
Fig. i. Density equilibrium sedimentation of Euglena chloroplast DNA in CsC1. a, 2/~g native DNA; b, 5/2g DNA denatured in o.13 ml 15 mM NaCI-I. 5 mlV[ sodium citrate adjusted to pH 12, io min, room temperature, chilled in ice and rapidly neutralized; c, 3/~g DNA renatured in o.13 ml, o.3o M NaCl-o.o3o M sodium citrate, p H 7, i h, 63°; d, 5 #g DNA denatured in o.13 ml 15 mM NaCI-I. 5 mM sodium citrate adjusted to pH 12, IO rain, room temperature. Centrifugation: Spinco Model E, An-D rotor, 44 77 ° rev./min, 18-2o h, 20°; tracings a, b, c are aligned with respect to the reference DNA peak, [15NJDNA of Pseudomonas aeruginosa (density z.742 g/cma). The densities in the neutral CsC1 gradients (a, b, c) are calculated relative to the reference according to SUEOKA19. The refractive indices of the neutral CsC] solution were kept in the range of 1.4ooo ±0.oo2. The densities of the samples in the alkaline gradient, d, were calculated as mentioned in the text. Fig. 2. Separation of single strands of Euglena chloroplast DNA. lO5/~g of Euglena chloroplast DNA were alkaline denatured, equilibrated in an alkaline CsC1 density gradient and fractionated as described in the text. The heavy and light fractions were pooled as indicated by the arrows. In the average of several such gradients the ratio of h e a v y to light single-strand DNA fraction was 4 to 3 and the overall recovery of DNA between 42 and 48 %. We isolated high and low density single-stranded chloroplast DNA in preparat i v e a m o u n t s u s i n g t h e m e t h o d of FLAMM el al. 13. Fig. 2 s h o w s t h e e l u t i o n p r o f i l e of s u c h a g r a d i e n t . Tile b a n d s a r e b r o a d a n d i n d i c a t e a d e n s i t y - p o p u l a t i o n of s t r a n d s o r s t r a n d f r a g m e n t s r a t h e r t h a n h o m o g e n o u s f r a c t i o n s of i n t a c t h e a v y a n d l i g h t s t r a n d s . T h e a r r o w s m a r k t h e r a n g e of t u b e s p o o l e d . I d e n t i c a l p e a k s f r o m s e v e r a l s u c h g r a d i e n t s w e r e c o m b i n e d a n d p r o c e s s e d a s m e n t i o n e d i n MATERIALS AND METHODS. Heavy and light fractions were tested in the analytical ultracentrifuge as shown i n Fig. 3- I n a s e r i e s of d e n a t u r i n g , r e n a t u r i n g a n d c o - r e n a t u r i n g e x p e r i m e n t s w e
Biochim. Biophys. Acta, 2o9 (197 ° ) 16-23
20
E. STUTZ, J. R. RAWSON
I
I
c
I
i
.
A
1.[
.
.
.
.
/)
oz
~o.~
L1.693 1.687
1.742
eiRNA
Fig. 3. Density equilibrium sedimentation of fractionated Euglena chloroplast DNA. a, 6/~g heavy fraction DNA, incubated in o.II ml 15 mlV[ NaCI-I.5 mM sodium citrate adjusted to pH 12, IO min, room temperature, chilled in ice, rapidly neutralized; b, 6/~g heavy fraction DNA incubated in o. 15 ml o.3o M NaCl-o.o3o M sodiumcitrate, pH 7, 6 h, 63°; c, 6/*g light fraction DNA, treated as mentioned sub a; d, 6 ktg light fraction DNA, treated as mentioned sub b; e, 3/~g heavy fraction plus 3/~g light fraction DNA in o.13 ml o.30 M NaCl-o.o3o M sodium citrate, pH 7, jointly incubated for 6 h, 63 °. Centrifugation, DNA reference and density calculation as mentioned in legend to Fig. i. Fig. 4. Saturation of chloroplast DNA filters with chloroplast ribosomal RNA. Filters loaded with 2o-2i/~g chloroplast DNA were challenged with increasing amounts of ribosomal RNA and incubated for 2o h at 64-65 `~in o.3o M NaCl-o.o3o ~ sodium citrate. t r i e d to e s t a b l i s h w h e t h e r a n d t o w h a t e x t e n t t h e h e a v y a n d light f r a c t i o n s c o n t a i n e d c o m p l e m e n t a r y s t r a n d s e g m e n t s . E a c h of t h e f r a c t i o n s w e r e d e n a t u r e d w i t h alkali, chilled a n d r a p i d l y n e u t r a l i z e d . W h e n a n a l y z e d in a n e u t r a l CsC1 d e n s i t y g r a d i e n t b o t h p r e p a r a t i o n s g a v e a r a t h e r b r o a d b a n d w i t h m e a n d e n s i t i e s of 1.7o 5 g / c m 3 (Fig. 3a) a n d 1.698 g / c m a (Fig. 3c), r e s p e c t i v e l y , t h e r e b y b r a c k e t i n g t h e n e u t r a l b u o y a n t d e n s i t y of d e n a t u r e d c h l o r o p l a s t D N A (Fig. I b ) . A l i q u o t s of t h e s a m e p r e p a r a t i o n s w e r e i n c u b a t e d for 6 h at 63 64 ° in 0.30 M N a C l - o . o 3 o M s o d i u m c i t r a t e a n d a g a i n a n a l y z e d in a n e u t r a l CsC1 g r a d i e n t . U n d e r s u c h r e n a t u r i n g c o n d i t i o n s t h e h e a v y f r a c t i o n s l i g h t l y c h a n g e d its o v e r a l l d e n s i t y as r e f l e c t e d in t h e r e s p e c t i v e t r a c i n g (Fig. 3b), s h o w i n g a s h o u l d e r on t h e left side. Also t h e l i g h t f r a c t i o n d e c r e a s e d its d e n s i t y s l i g h t l y u n d e r r e n a t u r i n g c o n d i t i o n s to a m e a n d e n s i t y of 1.693 g / c m 3 (Fig. Biochim. ldiophys. ,4cta, 209 (197 o) 16 23
E U G L E N A CHLOROPLAST SINGLE-STRAND
DNA
21
3d). But in both cases the bands remained broad, indicating that the majority of strand segments did not interact with each other. However, when equimolar amounts of the heavy and light fractions were combined and incubated under identical conditions, sharply banding DNA resulted with a density of 1.687 g/cm 3 (Fig. 3e). This value is close to the neutral buoyant density of native chloroplast DNA and identical with the value of renatured chloroplast DNA (see Fig. I). These data indicate that the heavy and light fractions represent populations of single-strand DNA which are essentially complementary to each other. In view of these results it became interesting to check whether there was any difference in the coding function between the two single-strand DNA fractions. We prepared highly purified Euglena chloroplast ribosomal RNA 1° which presumably is a product of the chloroplast genome. We hybridized it with total chloroplast DNA, to establish first the saturation value under a given set of incubation conditions (Fig. 4). Saturation is attained with 6-8/zg ribosomal RNA (22 S/I 7 S) per 2 o # g chloroplast DNA. The excess ribosomal RNA used to saturate 0.2 #g of DNA sites was 3o-4o-fold and below the 6o-fold excess needed under similar conditions in a bacterial system 15. Identical incubation conditions were used for hybridizing ribosomal RNA to fractionated chloroplast DNA (Table I). We saw that the heavy fraction
TABLE I H Y B R I D I Z A T I O N BETV*rEEN t?~UGLENA RIBOSOMAL
R N A AND VARIOUS T Y P E S OF E U G L E N A D N A
Specific a c t i v i t y , c o r r e c t e d for q u e n c h i n g : 145 c o u n t s / m i n pe r o . I / ~ g 22-S/I7-S r i b o s o m a l R N A
DNA source
Chloroplast heavy fraction
Chloroplast light fraction
DNA ~ug)
s2P-labeled ribosomal RNA source
RNA ~ug)
8 IO-I I
9-1o
Chloroplast 22 S/I 7 S Chloroplast 22 S/I 7 S
16 8 16
Chloroplast total
22
Chloroplast 22 S/I 7 S
8 16
Counts/rain per Jilter
Hybridization (%)
Charged
Blank
347 302 335 312
14 17 17 19
2.3 1.9 2.2 2.0
39 45 45 38
14 16 18 20
o.i 0.2 0.2 o.i
4o2 364 4o7 4o 7
25 21 19 21
1.2 i.o 1.2 1.2
retained approx. IO times more 32P-labeled ribosomal RNA than the light fraction (values corrected for blanks) and the percentage hybridization of the h e a v y fraction DNA is about twice the percentage value of total DNA. This indicates that the heavy DNA fraction contains the majority of base sequences coding for the 22-S/I7-S chloroplast ribosomal RNA. Biochim. B i o p h y s . ~tcta, 2o9 (197 ° ) 16-23
22
E. STUTZ, J. R. RAWSON
DISCUSSION
According to these results it is possible to split the Euglena chloroplast DNA in an alkaline CsC1 density gradient into two populations of single-strand DNA which differ in their overall alkaline buoyant density by o.o23 g/cm s. VINOGRAD eb al. 17 have shown that the buoyant density of denatured single-strand DNA sharply increases in the range of p H 12 as a function of the T + G content. They therefore suggested this procedure to discriminate between complementary strands of unequal base composition. The method proved feasible in case of relatively small and homogenous genomes as e.g., phage DNA is or mouse satellite nuclear DNA 13 where individual heavy and light strands could be separated. The Euglena chloroplast genome has a molecular weight of approx. 6.2. lO9 (ref. I) and is easily fragmented during the isolation. We would, therefore, expect that each of the two single-strand DNA fractions we have isolated contain strand fragments from both in vivo chromosomal strands. Comparing the G + C content of chloroplast ribosomal RNA which is 52 % (ref. IO) with the G + C content of chloroplast DNA which is 25 %, one realizes that the ribosomal RNA cistrons are quite different from the overall DNA base composition. In fact, the chances for the ribosomal RNA cistrons ( G + T = 45 ~'o) to band in the heavier of the two alkaline single-strand DNA fractions ( G + T -- 57 °/o) are marginal unlessT- or G-rich sequences are contiguous to the cistrons. If such additional sequences are removed because of strand fragmentation the cistrons would tend to band in the light alkaline single-strand DNA fraction. Such m a y be the reason for the IO ~o transcribing base sequences found in the light fraction. Under saturation conditions 1.2 }"o of the chloroplast DNA hybridizes with 22-S/I7-S RNA. This is close to the I °/o reported by SCOTT AND SMILLIEg0. We know from competition experiments that the annealing reaction is specific. On this basis and taking the size of the Euglena DNA as 6.2.Io 9 (tool. wt.) the number of cistrons coding for 22-S/I7-S RNA is about twenty. Each of the two isolated single-strand DNA preparations displays a certain renaturing capacity as reflected by the reverse shift in neutral buoyant density. This m a y be due to incomplete separation of the complementary strand fragments and/or intra-strand annealing as was, e.g., shown for mouse single-strand satellite DNA 5. Nothing is known at present about regional differences in base composition or possible secondary structure formation in single-strand chloroplast DNA. The quality of the alkaline separated single-strand DNA fraction might, therefore, change with the fragment size of the DNA preparation. BORST AND AAIJ 7 have shown that rat liver mitochondrial DNA undergoes strand separation in an alkaline CsC1 density gradient and the heavy strand acts as exclusive messenger strand under the chosen experimental conditions. OISHIs has reported that Bacillus subtilis heavy strand DNA which was isolated by using the methylated albumin kieselguhr column 21 hybridizes with ribosomal and transfer RNA exclusively. He concludes combining these data with earlier results from KUBINSKY et al. ~2 that all species of B. subtilis RNA are complementary to the heavy strand segments. It remains to be shown whether the heavy fraction of Euglena chloroplast DNA also codes for all species of RNA.
Biochim. Biophy$. Acta, 209 (197 o) 16--23
EUGLENA CHLOROPLAST SINGLE-STRAND
DNA
23
ACKNOWLEDGMENTS
The Pseudomonas aeruginosa was a gift from Dr. N. Welker. We thank Mrs. K. Patel for skillful technical help. This investigation was supported by Biomedical Science Support Grant FR-o7o28 and a Predoctoral Fellowship I-FI-GI7-3774-o2 to J. R. R. from the National Institutes of Health. We are grateful to Dr. H. Null for additional support. REFERENCES I G. BRAWERMANN AND J. ~v[. EISENSTADT, Biochim. Biophys. Acta, 91 (1964) 477. 2 D. S. RAY AND P. C. HANAWALT, J. Mol. Biol., 9 (1964) 812. 3 M. EDELMAN, C. A. COWAN, H. T. EPSTEIN AND J. A. SCHIFF, Proc. Natl. Acad. Sci. U.S., 52 /I964) 1214. 4 G. CORNED, L. ZARDI AND E. POLLI, J. Mol. Biol., 36 (1968) 419 . .5 W. G. FLAMM, P. ])¢[. B. WKLKER AND M. McCALLUM, J. Mol. Biol., 4 ° (1969) 423 . 6 G. CORNED, E. GINELLI AND E. POLLI, J. Mol. Biol., 33 (1968) 331. 7 P. BURST AND C. AAIJ, Biochem. Biophys. Res. Commun., 34 (1969) 358. 8 M. OISHI, Proc. Natl. Acad. Sci. U.S., 62 (1969) 256. 9 J. R. RAWSON AND E. STUTZ, J. Mol. Biol., 33 (1968) 309. io J. R. RAWSON AND E. STUTZ, Biochim. Biophys. Acta, 19o (1969) 368. i i J. ~¢[ARI~IUR, J. Mol. Biol., 3 (1961) 208. 12 W. G. FLAMM, H. E. BOND AND H. E. BURR, Biochim. Biophys. Acta, 129 (1966) 31o. 13 W. G. FLAMM, IV[.•CCALLUM AND P. M. B. WALKER, Proc. Natl. Acad. Sci. U.S., 57 (1967) 1729 14 M. CRAMER AND J. MEYERS, Arch. Mikrobiol., 17 (1952) 384 . 15 D. GILLESPIE AND S. SPIEGELMAN, J. Mol. Biol., 12 (1965) 829. 16 J. VINOGRAD AND H. E. HEARST, Progr. Chem. Org. Nat. Prod., 20 (1962) 395. 17 J. VINOGRAD, J. MORRIS, N. DAVlDSON AND N. DOVE, Proc. Natl. Acad. Sci. U.S., 49 (1963) 12. 18 W. DOERFLER AND D. S. HOGNESS, J. Mol. Biol., 33 (1968) 635. 19 N. SUEOKA, J. Mol. Biol., 3 (1961) 31. 20 N. S. SCOTT AND R. N. SMILLIE, Biochem. Biophys. Res. Commun., 28 (1967) 598. 21 14. RunNER, J. D. KARKAS AND E. CHARGAFF, Proc. Natl. Acad. Sci. U.S., 60 (1968) 630. 22 H. I~UBINSKY, Z. OPARA-KUBINSKA AND W. SZYBALSKY, J. Mol. Biol., 20 (1966) 313 .
Biochim. Biophys. Acta, 2o9 (197 o) 16-23