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Chromatographic purification of one of the complementary strands of DNA of phage α
SHORT COMMUNICATIONS
~, Biochim. Biophys. Acta, 71 (1963) 45 Nature, 200 (1963) 148. LLER, J. Biol. Chem., 224 (1957) 13. AND P . C. ZAMECNIK, J. B i o l . Chem., 2 J. Org. Chem., 17 (1952) 1298. , A . L . F A R R AND R . J . R A N D A L L , J . B
3 (1951)
Am. Chem. Soc., 56 (1934) 658.
3 Biochim. Biophys. Ach
5IO--514
SC 7131 romatographic purification of one of the comple )lementa~ of phage a*
r DNA
aperate ~vious reports have described the unusual p h y s i c a l behaviou~ ige a, as well as its main physico-chemical and structural stru as tracenshowed ugal analysis in CsC1 density gradients of this DNA aft after heat d~ presence of two distinct bands, indicated as ax and 0t2, with f 1.712 de each )ectively. These b 711-1.713) and 1.72o (1.719-1.722) g/cm 3, respectively abation ter with respect to width and absorbancy and they disappe~ ear the LII, 50°, giving rise to a single band with an average t.X~ll~lLy densil UJL density of JL.//lJt)) 1.7(~ YVILU.,IL1~ II~.~OLL lsity of the band formed b y the same native DNA (1.7o4) (I.7¢ (Fig. 2b). For this reason, flemen)resent thq the two separated complementhe ',~i c(, and ~2 bands have been considered to represent 'ical y strands 3-5. Their further characterization, as to phy ,sico-chemical and biological tar~ )hic account of the chromatographi( ~perties, required their preparative separation. An acc( prol0 important experiments on the separation ~aration of ~i and c¢2 has been communicate& and irn However, the chromatographic work work ands thus separated have been reported s-~°. However strands aaviour has; not yet been described in detail b y the authors. The chromatographic behaviom th( of denatured x-DNA and in particular the purification of xl are the subject of the present paper. The investigated DNAA was prepared according to the procedure described byY ~ht S I N S H E I M E R 11 and consisted d of a homogeneous material with high molecular weighl in ttion was brought about b y heating at 95 ° for IO min it of about 35" lO6. Denaturation enum citrate (which is referred to as the standard conceno.15 M NaC1, o.o15 M sodium ~g") tration) and quickly cooling. Thermal treatment for renaturation ("annealing" -citrate ring DNA (15-2o #g/ml) for 4-8 h at 60 ° in saline--citrat( was carried out by incubatin collard concentration. Material was chromatographed on col solution of twice the standard umns of kieselguhr coatedl with methylated albumin, using the procedures of MAN ve DELL AND H E R S H E Y la and] of SUEOKA AND CHENGis. Both these procedures gav~ ed ,qution of native ~-DNA. On the other hand, with denature( successful adsorption and elution ~-DNA, chromatographed in the manner of ref. 13, the results were not satisfactor3.y arched, with regard to recovery, separation of peaks and reproducibility. We searched nd Lblecon( conditions, therefore, for more favourable onditions by varying the pH, and the composition an( * P a r t of t h i s w o r k w a s c o m m u nmi c a t e d to t h e ISt Convegno I t a l i a n o di Biofisica, R o m e , Juunnee, 1963 .
Biochim. Biophys. Acta, 80 (1964) 514-5116
ORT C O M M U N I C A T I O N S
ras found that 5 o ~ o % of the ads( can be ous concentrations of buffer and I 1at the ng the p H to I I . In a series of pik zts the elution with high resolution were given "adient 3sphate buffer (pH 7.9). Thus eluti e rise to a pattern revealing three ments; component eluted in the range 0.3.' [icated bsence of contaminating a2 was con camin. On the other hand the same test on the ion to degrees, indicatin .t iJx~aJXO x~v~o.xx~xx .tx.iio~t.uxo~g~vax t.,J various v~xa~o u ~x~o, nce of fr( n varying amounts. Similar observations on the materials ma experiits (using both gradient and stepwise elution) indicated indica that, )m the distripeak (purified mz), there is a gradual superposition,of c%. Be( ion of the two strands in the elution pattern, 0q can be h found i y only he first peak. The evident difficulty of this separati )aration contr le high [ability encountered in these experiments. This variability vaE due to ontrnlled factors and affected principally the recovery recov educed of E lowing ecular weight of purified 0q, which sediments with 13 S whe ks and denatured ~t-DNA sediment with 25 S, appear= )ears to be a tor for iration. ned b y Stepwise elution was then employed, in correspondel )ondence to the salil ,lution, :liente]ution. Samples of 8oo--llOOFg (15 Fg/ml, in in standard stay onto a column of 3 cm ;m :ected to 0.25 M phosphate buffer (pH 7"9)) were loaded loa equilibrated with ,meter) b y 1.5 cm (height), thermostated at 25 °, and previously I 2 60 ZO.S) o~ t,. 40
127.71
Q.
19.81 20
g
17.31
17.11
14.91
16.21
0.375 4
0.400 4
0.500 4
11o.8)
F'LUENTS F" FRACTIONS 13
0.300 4
0.325 5
0.350 4
pH 11,6 8
Fig. I. C h r o m a t o g r a p h y of d e nna t u r e d D N A f r o m p h a g e ~. T h e h i s t o g r a m r e p r e s e n t s t h e p)eaks eaks a n d # g of D N A p e r fraction (average), verage), e l u t e d b y t h e i n d i c a t e d NaCl m o l a r i t i e s a n d b y t h e p H - i I buffer. T h e v a l u e s in b r a c k e t ss indicate t h e c o r r e s p o n d i n g r e c o v e r y of D N A in e a c h p e a k (as As percentag, :enrages of t h e t o t a l a d s o r b e d D N A ) .
the same solvent. Elution was vas carried at a flow rate of about 2 ml/min, with continuontinuous recording of effluent absor Lbsorption at 254 m/z A typical experiment from a lon~g series is illustrated in Fig. I. When these peaks were examined after heating at 6o °, ° the peak eluted at o.3o M gave rise to a single band with a density of 1.711, correrresponding to 0q (Fig. 2, a and .nd b). The potential efficiency of this procedure for pre)reparing substantial amounts; of ~1 in high purity is indicated b y quantitative evaluatior .luation of a group of ten experiments .'nts, in which the experimental conditions and the result~ alts (including ultracentrifugal analysis after annealing) were similar to those reportec ~orted above. In the peak consistin ing of purified al the recovery (as percentage of the totad adsorbed D r ' A ) was I 4 ~ 2 (average+S.E.). Thus about 3 ° % of ~1 (i.e. lOO-15o/~g ~g) Biochim.
Biophys.
Acta,
;16 8o (I964) 514-511
HORT COMMUNICATIONS
El
L],/ / /
I
1.704
m)
| 1.711
• 2. U ] t r a c e n t r i f u g a [ analysis, in CsC| d e n s i t y - g r a d i e n t , a. The T peak e n the e x p e r i m e n t in Fig. i. The reference b a n d is g i v e n by native nal DNA t reference bm n phage ~: I, native; II, denatured; III, renatured. The ret D N A from the h o s t (B. megatheriu 'atherium).
M NaC1 b. D N A V native
obtained in high purity from a single chromatogra ~raphic pro( fled ~x Despite the above-mentioned variability, sufficient sufficiel amou: re been prepared by this procedure, to investigate the role of a late for [A synthesis 8. for their helpful and We wish to thank Professors M. AGENO and F. GRAZIOSI GR and Mr. E. I~ATTOCCIA TOCCIA stimulatin nulating interest in this work, as well as Mr. G. CONTI Co for invaluable technical assistance.
;tituto Superiore di SanitY, Rome Istituto and Laboratorio Internazionale di Genetica e Bio/isica, Naples (Italy)
S.
AURISICCHI0 ~ICCHIO
E . DORE C. FRONTALI F.
GAETA
G. T o s c m td., 1 A . CELANO, S. AURISICCHIO), A. CoPPO, P. DONINI AND F. CrRAZlOSI, NUOVO Cimento Suppl. I8 (196o) I9O. )TTO, A . CoPPO, P . DONINI, C. FRONTALI AND F . GRAZIOSI, Nuovo NuOVC S. AURISICCHIO, A. CHIOZZOTTO
Cimento Suppl., 18 (196o) 197. de~ a S. AURISICCHIO, A . CoPPO, PP.. DONINI, C. FRONTALI, F. GRAZlOSI AND G. TOSCHI, Rapporti dei Laboratori di Fisica dell'Instituto ~tituto Superiore di SanitY, ISS 61/33, J u n e 1961. 4 S. CORDES, J . MARMUR AND) H. T. EPSTEIN, Nature, 19I (1961) lO97. S S. AURISICCHIO, C. FRONTALI A, F. GRAZIOSI AND G. TOSCHI, Nuovo Cimento suppl., 25 (1962) 35 35Mol g W . FULLER, M. SPENCER, A~. . DANIELE SARGENTINI, F. SGARLATA AND R . LANGRIDGE, J. Mol.
Biol., 6 (I963) 5IO. 7 S. CORDES, H. T. EPSTEIN AND AND J. M.ARMUR, Federation Proc., 21 (1962) 375. WEIS.¢ a G. P . TOCCHINI-VALENTINI, 72~'. STODOLSKY, i~. SARNAT, S. AURISlCCHIO, F. C*RAZIOSI, S. WEISS AND E . P . CrEIDUSCHEK, Proc. "oc. Natl. Acad. Sci. U.S., 5° (1963) 935.
9 j. MARMUR, Cold Spring Harbour arbour Syrup. Q,uant. Biol., in the press. j, MARMUR AND S. CORDES , In/ormational Macromolecules, Academic Press, New York, 19633, P. 79. n R. L. SINSHEIMER, J. Mol. Biol., i (1959) 431, j. D. MANDEL AND A . D . HERSHEY tERSHEY, Anal. Biochem., I (196o) 66. 13 N . SUEOKA AND T. Y . CHENG SG, J. Mol. Biol., 4 (1962) 161.
10
Received December 2nd, 1963 Biochim. Biophys. Acta, 80 (1964) 514-516 514-51(
~, Biochim. Biophys. Acta, 71 (1963) 45 Nature, 200 (1963) 148. LLER, J. Biol. Chem., 224 (1957) 13. AND P . C. ZAMECNIK, J. B i o l . Chem., 2 J. Org. Chem., 17 (1952) 1298. , A . L . F A R R AND R . J . R A N D A L L , J . B
3 (1951)
Am. Chem. Soc., 56 (1934) 658.
3 Biochim. Biophys. Ach
5IO--514
SC 7131 romatographic purification of one of the comple )lementa~ of phage a*
r DNA
aperate ~vious reports have described the unusual p h y s i c a l behaviou~ ige a, as well as its main physico-chemical and structural stru as tracenshowed ugal analysis in CsC1 density gradients of this DNA aft after heat d~ presence of two distinct bands, indicated as ax and 0t2, with f 1.712 de each )ectively. These b 711-1.713) and 1.72o (1.719-1.722) g/cm 3, respectively abation ter with respect to width and absorbancy and they disappe~ ear the LII, 50°, giving rise to a single band with an average t.X~ll~lLy densil UJL density of JL.//lJt)) 1.7(~ YVILU.,IL1~ II~.~OLL lsity of the band formed b y the same native DNA (1.7o4) (I.7¢ (Fig. 2b). For this reason, flemen)resent thq the two separated complementhe ',~i c(, and ~2 bands have been considered to represent 'ical y strands 3-5. Their further characterization, as to phy ,sico-chemical and biological tar~ )hic account of the chromatographi( ~perties, required their preparative separation. An acc( prol0 important experiments on the separation ~aration of ~i and c¢2 has been communicate& and irn However, the chromatographic work work ands thus separated have been reported s-~°. However strands aaviour has; not yet been described in detail b y the authors. The chromatographic behaviom th( of denatured x-DNA and in particular the purification of xl are the subject of the present paper. The investigated DNAA was prepared according to the procedure described byY ~ht S I N S H E I M E R 11 and consisted d of a homogeneous material with high molecular weighl in ttion was brought about b y heating at 95 ° for IO min it of about 35" lO6. Denaturation enum citrate (which is referred to as the standard conceno.15 M NaC1, o.o15 M sodium ~g") tration) and quickly cooling. Thermal treatment for renaturation ("annealing" -citrate ring DNA (15-2o #g/ml) for 4-8 h at 60 ° in saline--citrat( was carried out by incubatin collard concentration. Material was chromatographed on col solution of twice the standard umns of kieselguhr coatedl with methylated albumin, using the procedures of MAN ve DELL AND H E R S H E Y la and] of SUEOKA AND CHENGis. Both these procedures gav~ ed ,qution of native ~-DNA. On the other hand, with denature( successful adsorption and elution ~-DNA, chromatographed in the manner of ref. 13, the results were not satisfactor3.y arched, with regard to recovery, separation of peaks and reproducibility. We searched nd Lblecon( conditions, therefore, for more favourable onditions by varying the pH, and the composition an( * P a r t of t h i s w o r k w a s c o m m u nmi c a t e d to t h e ISt Convegno I t a l i a n o di Biofisica, R o m e , Juunnee, 1963 .
Biochim. Biophys. Acta, 80 (1964) 514-5116
ORT C O M M U N I C A T I O N S
ras found that 5 o ~ o % of the ads( can be ous concentrations of buffer and I 1at the ng the p H to I I . In a series of pik zts the elution with high resolution were given "adient 3sphate buffer (pH 7.9). Thus eluti e rise to a pattern revealing three ments; component eluted in the range 0.3.' [icated bsence of contaminating a2 was con camin. On the other hand the same test on the ion to degrees, indicatin .t iJx~aJXO x~v~o.xx~xx .tx.iio~t.uxo~g~vax t.,J various v~xa~o u ~x~o, nce of fr( n varying amounts. Similar observations on the materials ma experiits (using both gradient and stepwise elution) indicated indica that, )m the distripeak (purified mz), there is a gradual superposition,of c%. Be( ion of the two strands in the elution pattern, 0q can be h found i y only he first peak. The evident difficulty of this separati )aration contr le high [ability encountered in these experiments. This variability vaE due to ontrnlled factors and affected principally the recovery recov educed of E lowing ecular weight of purified 0q, which sediments with 13 S whe ks and denatured ~t-DNA sediment with 25 S, appear= )ears to be a tor for iration. ned b y Stepwise elution was then employed, in correspondel )ondence to the salil ,lution, :liente]ution. Samples of 8oo--llOOFg (15 Fg/ml, in in standard stay onto a column of 3 cm ;m :ected to 0.25 M phosphate buffer (pH 7"9)) were loaded loa equilibrated with ,meter) b y 1.5 cm (height), thermostated at 25 °, and previously I 2 60 ZO.S) o~ t,. 40
127.71
Q.
19.81 20
g
17.31
17.11
14.91
16.21
0.375 4
0.400 4
0.500 4
11o.8)
F'LUENTS F" FRACTIONS 13
0.300 4
0.325 5
0.350 4
pH 11,6 8
Fig. I. C h r o m a t o g r a p h y of d e nna t u r e d D N A f r o m p h a g e ~. T h e h i s t o g r a m r e p r e s e n t s t h e p)eaks eaks a n d # g of D N A p e r fraction (average), verage), e l u t e d b y t h e i n d i c a t e d NaCl m o l a r i t i e s a n d b y t h e p H - i I buffer. T h e v a l u e s in b r a c k e t ss indicate t h e c o r r e s p o n d i n g r e c o v e r y of D N A in e a c h p e a k (as As percentag, :enrages of t h e t o t a l a d s o r b e d D N A ) .
the same solvent. Elution was vas carried at a flow rate of about 2 ml/min, with continuontinuous recording of effluent absor Lbsorption at 254 m/z A typical experiment from a lon~g series is illustrated in Fig. I. When these peaks were examined after heating at 6o °, ° the peak eluted at o.3o M gave rise to a single band with a density of 1.711, correrresponding to 0q (Fig. 2, a and .nd b). The potential efficiency of this procedure for pre)reparing substantial amounts; of ~1 in high purity is indicated b y quantitative evaluatior .luation of a group of ten experiments .'nts, in which the experimental conditions and the result~ alts (including ultracentrifugal analysis after annealing) were similar to those reportec ~orted above. In the peak consistin ing of purified al the recovery (as percentage of the totad adsorbed D r ' A ) was I 4 ~ 2 (average+S.E.). Thus about 3 ° % of ~1 (i.e. lOO-15o/~g ~g) Biochim.
Biophys.
Acta,
;16 8o (I964) 514-511
HORT COMMUNICATIONS
El
L],/ / /
I
1.704
m)
| 1.711
• 2. U ] t r a c e n t r i f u g a [ analysis, in CsC| d e n s i t y - g r a d i e n t , a. The T peak e n the e x p e r i m e n t in Fig. i. The reference b a n d is g i v e n by native nal DNA t reference bm n phage ~: I, native; II, denatured; III, renatured. The ret D N A from the h o s t (B. megatheriu 'atherium).
M NaC1 b. D N A V native
obtained in high purity from a single chromatogra ~raphic pro( fled ~x Despite the above-mentioned variability, sufficient sufficiel amou: re been prepared by this procedure, to investigate the role of a late for [A synthesis 8. for their helpful and We wish to thank Professors M. AGENO and F. GRAZIOSI GR and Mr. E. I~ATTOCCIA TOCCIA stimulatin nulating interest in this work, as well as Mr. G. CONTI Co for invaluable technical assistance.
;tituto Superiore di SanitY, Rome Istituto and Laboratorio Internazionale di Genetica e Bio/isica, Naples (Italy)
S.
AURISICCHI0 ~ICCHIO
E . DORE C. FRONTALI F.
GAETA
G. T o s c m td., 1 A . CELANO, S. AURISICCHIO), A. CoPPO, P. DONINI AND F. CrRAZlOSI, NUOVO Cimento Suppl. I8 (196o) I9O. )TTO, A . CoPPO, P . DONINI, C. FRONTALI AND F . GRAZIOSI, Nuovo NuOVC S. AURISICCHIO, A. CHIOZZOTTO
Cimento Suppl., 18 (196o) 197. de~ a S. AURISICCHIO, A . CoPPO, PP.. DONINI, C. FRONTALI, F. GRAZlOSI AND G. TOSCHI, Rapporti dei Laboratori di Fisica dell'Instituto ~tituto Superiore di SanitY, ISS 61/33, J u n e 1961. 4 S. CORDES, J . MARMUR AND) H. T. EPSTEIN, Nature, 19I (1961) lO97. S S. AURISICCHIO, C. FRONTALI A, F. GRAZIOSI AND G. TOSCHI, Nuovo Cimento suppl., 25 (1962) 35 35Mol g W . FULLER, M. SPENCER, A~. . DANIELE SARGENTINI, F. SGARLATA AND R . LANGRIDGE, J. Mol.
Biol., 6 (I963) 5IO. 7 S. CORDES, H. T. EPSTEIN AND AND J. M.ARMUR, Federation Proc., 21 (1962) 375. WEIS.¢ a G. P . TOCCHINI-VALENTINI, 72~'. STODOLSKY, i~. SARNAT, S. AURISlCCHIO, F. C*RAZIOSI, S. WEISS AND E . P . CrEIDUSCHEK, Proc. "oc. Natl. Acad. Sci. U.S., 5° (1963) 935.
9 j. MARMUR, Cold Spring Harbour arbour Syrup. Q,uant. Biol., in the press. j, MARMUR AND S. CORDES , In/ormational Macromolecules, Academic Press, New York, 19633, P. 79. n R. L. SINSHEIMER, J. Mol. Biol., i (1959) 431, j. D. MANDEL AND A . D . HERSHEY tERSHEY, Anal. Biochem., I (196o) 66. 13 N . SUEOKA AND T. Y . CHENG SG, J. Mol. Biol., 4 (1962) 161.
10
Received December 2nd, 1963 Biochim. Biophys. Acta, 80 (1964) 514-516 514-51(