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Fractionation of DNA I. Countercurrent distribution of native and degraded DNA
62 4
SHORT COMMUNICATIONS
increased if labeled compounds are used and the detection is carried out by autoradiography. I wish to thank Dr. P. C. ZAMECNIK and Dr. H. M. KALCKARfor their generous support during the course of this work. This investigation was supported by grant 3 TE CA 5o18 from the U.S. Public Health Service. This is publication No. 114o of the Cancer Commission of Harvard University.
The John Collins Warren Laboratories O~ the Huntington Memorial Hospital o/ Harvard University, Massachusetts General Hospital, Boston, Mass., U.S.A.
KURT RANDERATH
Naturwissenscha[ten, 39 (1952) 86. p. REICHARD, Acta Chem. Scand., 12 (1958) 2048. J. X. KHYM AND W. E. COHN, Biochim. Biophys. dcta, 15 (1954) 139. p. REICHARD, A. ]~ALDESTEN AND L. I~UTBERG, J. Biol. Chem., 236 (1961) 115o. p. REICHARD, J. Biol. Chem., 237 (1962) 3513. S. S. COHEN, H. D. BARNER AND J. LICHTENSTEIN, J. Biol. Chem., 236 (1961) 1448. H, G. KLEMPERER, J. S. KRAKOW A.ND E. S. CANELLAKIS, Biochim. Biophys. Acta, 61 (1962) 43. K. I~ANDERATH,Biochim. Biophys. Acta, 61 (1962) 852. it K. t{.ANDERATH, Thin-layer Chromatography, Verlag C h e m i e VVeinheim a n d e r B e r g s t r a s s e , a n d A c a d e m i c Press, N e w York, 1963 . 10 j . X. KHYM, L. P. ZILL AND W. E. COHN,in C. CALMON AND T. R. E. I'{RESSMAN, Ion-Exchangers in Organic and Biochemistry, Interscience, N e w York, 1957, p. 392. 11 W. E. COHN AND V. J. ]3OLLUM, Biochim. Biophys. Acta, 48 (1961) 588. z L. JAENICKE AND I. VOLLBRECHTSHAUSEN,
z it 4 5 e '/ 8
Received July ISt, 1963 Biochim. Biophys. Acta, 76 (1963) 622-624 SC 7114
Froctionotion of D N A I. Countercurrent distribution of notive ond degraded D N A Heterogeneity of DNA molecules from a given tissue has been examined by a number of methods including ultracentrifuga] sedimentation analysis and helix-coil transition during thermal denaturation 1, and ion-exchange chromatography *. 1RNA has been fractionated by countercurrent distribution in a number of 2-phase systems according to base composition3, 4 and countercurrent distribution has also been applied to the fractionation of soluble RNA's 5. Preliminary studies e with countercurrent distribution of degraded commercial I)NA from calf thymus achieved fractionation dependent upon base composition but undenatured I)NA remained largely in the aqueous phase and was not fractionated by these methods. In the present studies it has been possible to fractionate DNA's from various sources by countercurrent distribution. I)NA was prepared from rat liver, Drosophila melanogaster and Escherichia coli by modifications of the 4-aminosalicylate-phenol method 7. Commercial I)NA from salmon sperm (Mann, "highly polymerized") was reprecipitated from aqueous solution with ethoxyethanol in the presence of 4 % lithium acetate. Degradation was achieved enzymically in some experiments by treating a 0.2 % solution of DNA Biochim. Biophys. Acta, 76 (1963) 624 627
625
SHORT COMMUNICATIONS
containing o.ooi M magnesium acetate with IO mg DNAase (EC 3.1.4.5) per ml for 60 min at 20 ° and in other experiments by heating for 4 ° min at IOO° a o.I % solution of DNA containing o.ooi M sodium acetate and IO % phenol. The degraded DNA was precipitated with ethanol in the presence of 4 % lithium acetate. DNA degraded by heating in this way was found to have an S2o of 3.5-4.0 S as determined in the Spinco model E ultracentrifuge. Solvent systems for countercurrent distribution were similar to those developed for RNA 4 except that lithium citrate was added in place of potassium citrate in the aqueous phase, lithium salts being found to give more satisfactory partition of I)NA than potassium salts. The proportions in the countercurrent distribution system best suited to I)NA separation were: organic mixture, 28 volumes; amine solution, 12 volumes; water, 39.5 volumes; 0.033 M lithium citrate, 12.5 volumes. This system was designated as solvent system 15o/12.5. The lithium ion concentration was critical since in a 15o/13.o system the majority of the degraded I)NA remained in the first 20 tubes during 80 transfers while in a 15o/12.o system the majority travelled with the organic phase. Native I)NA showed a similar dependence on the lithium ion concentration though less marked in degree. 5-15 mg degraded I)NA or 1-'2 mg native ])NA were distributed in this solvent system (2 ml each phase) over 80 transfers. The I)NA was first dissolved in 2 ml of each phase of a 15012 system and before transferring the I)NA into the lower phase of the 15o/12.5 solvent system it was passed through a gradient of increasing concentration of lithium ion in single tube steps of solvent systems 15o/6, 15o/8, 15O/lO. This permitted optimal solubilization of I)NA in the organic phase (15o/12.5 system). After 80 transfers ethanol (I ml) was added to the contents of each tube to make one phase and the absorption read at 260 m F. For determination of base composition of denatured DNA fractions, 15 mg were used in countercurrent distribution and the contents of each lO-2O tubes pooled to give a small number of fractions. The DNA was recovered4 and base ratios s determined as described previously. Fig. I shows the patterns of distribution over 80 transfers of heat-degraded I)NA
.2•0
A260 1.5
!.i 1.C
•"•..: I
"'.••
•..:"-
0.~
2o 3'o
40
50
6O
70
8O
TPonsfer number
Fig. I. C o u n t e r c u r r e n t distribution p a t t e r n s of heat-degraded D N A ' s over 80 transfers in solvent s y s t e m I5O[I2. 5. D N A from: r a t liver ( - - - ) , s a l m o n s p e r m ( . . . ) , and E. coli ( ).
Biochim. Biophys. Acta, 76 (1963) 624-627
626
SHORT COMMUNICATIONS
from rat liver, salmon sperm and E. coli. A degree of coincidence exists between some peaks for DNA from rat liver and salmon sperm but in general marked differences exist among the 3 species illustrated. Reproducibility of pattern was good for DNA from the same tissue and species under the given conditions of degradation. As summarized in Table I this distribution shows trends in base composition similar to TABLE BASE
COMPOSITIONS
OF D E G R A D E D
DNA
BY COUNTERCURRENT
I FROM
RAT LIVER
FRACTIONATED
DISTRIBUTION
F r a c t i o n s are n u m b e r e d from the a q u e o u s end of the c o u n t e r c u r r e n t distribution. Heat-degraded D N A was fractionate~t in solvent s y s t e m i5o as described in the text, DNAase-degraded D N A in solvent s y s t e m i2 7 (see ref. 3). (Fractions I and 2 represented unfractionated D N A and have been omitted here.) Moles base/zoo moles Treatment
Fraction
Heat
I 2 3 4 3 4 5 6 7
DNAase
Guanine
Adenine
Cytosine
Thymine
24.9 26.0 21.5 16.3 29.4 27. 5 26.2 22.2 18.o
25.3 28.8 30.8 3 o.8 24. 9 26.2 29.4 32.1 32.9
24.9 20.8 18.7 20.5 22.2 20. 4 19.4 17. 7 19.1
24.9 24. 4 29.0 32.4 23. 5 25.9 25.0 28.0 3o.0
those found for RNA4 and for commercial DNA from calf thymus in other solvent systems e. DNA segments with a higher proportion of guanine and cytosine remained at the aqueous end while those with a higher proportion of adenine and thymine travelled further in the organic phase. Native DNA's (Fig. 2) showed a similar spread on countercurrent distribution in the I5O/I2. 5 solvent system but very different profiles from those of denatured
o.3t A26o
0.2
0.1 °
,
20
30 4Q 5Q £:K3 TFQRSfeP number
70
80
Fig. 2. C o u n t e r c u r r e n t distribution p a t t e r n s of native D N A ' s over 80 transfers in solvent s y s t e m I5O/I2. 5. D N A from: r a t liver ( - - - ) , D. melanogaster ( ), and E. coli (...).
Biochim. Biophys. Acta, 76 (1963) 624-6"27
SHORT COMMUNICATIONS
627
DNA's. For different preparations from the same tissue and species, reproducibility of profile was good with respect to general pattern but some quantitative variation occurred in individual peaks. This could be due to the small amount of native DNA which could be employed. Since insufficient material can be recovered from the countercurrent distribution to permit direct determination of base ratios of fractions, the basis of this fractionation of native DNA is not yet clear and other approaches to this problem are presently being studied. The fractionation of both native and degraded I)NA b y countercurrent distribution methods suggests considerable usefulness for this technique in the study of both base sequences and biological activities of DNA. We have found transforming activity to be retained b y native DNA from Bacillus subtilis after treatment with the 15o solvent system and this fact together with the degree of fractionation obtained with native DNA indicate the possible application of countercurrent distribution to separation of genetic functions of I)NA molecules. We wish to thank Mr. J. R. B. HASTINGS for the preparation of DNA from Drosophila melanogaster, Dr. M. GUEST for the preparation of DNA from Escherichia coli, Dr. A. D. Vlzoso for sedimentation measurements, and Professor A. HADDOW, F.R.S., for his interest in this work. This investigation has been supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research: Royal Cancer Hospital) from the Medical Research Council, the British Empire Cancer Campaign, the Anna Fuller Fund and the National Cancer Institute of the National Institutes of Health, U.S. Public Health Service. One of us (C.K.) is Arthur A. Thomas Cancer Research Fellow of the AntiCancer Council of Victoria, on leave from the Baker Medical Research Institute, Melbourne. Chester Beatty Research Institute, Institute o/ Cancer Research: Royal Cancer Hospital, F u l h a m Road, London (Great Britain)
CHEVKIDSON K. S. KIRBY
1 p. DOTY,J, MARMURAND 1~. SUEOKA,Broohhaven Syrup. Biol., 12 (1959) I. 2 H. S. I~.OSENKRANZANDA. BENDICH,J. Am. Chem. Soc., 81 (1959) 9o2. * K. S. KIRBY, Biochim. Biophys. Acta, 61 (1962) 5o6. 4 K. S. KIRBY,J. R. B. HASTINGSANDM. A. O'SULLIVAN,Biochim. Biophys. Acta, 61 (1962) 978. R. W. HOLLEY,J. APGARA.NDB. P. DOCTOR,Ann. N.Y. Acad. Sci., 88 (196o) 745. e K. S. KIRBY, The Molecular Basis o/Neoplasia, Texas Univ. Press, 1962, p. 59. K. S. KIRBY. Biochim. Biophys. Acta, 36 (1959) 117. s K. S. KIRBY, Biochem. J., 66 (1957) 495. Received J u l y 4th, 1963 Biochim. Biophys. Aaa, 76 (1963) 624-627
SHORT COMMUNICATIONS
increased if labeled compounds are used and the detection is carried out by autoradiography. I wish to thank Dr. P. C. ZAMECNIK and Dr. H. M. KALCKARfor their generous support during the course of this work. This investigation was supported by grant 3 TE CA 5o18 from the U.S. Public Health Service. This is publication No. 114o of the Cancer Commission of Harvard University.
The John Collins Warren Laboratories O~ the Huntington Memorial Hospital o/ Harvard University, Massachusetts General Hospital, Boston, Mass., U.S.A.
KURT RANDERATH
Naturwissenscha[ten, 39 (1952) 86. p. REICHARD, Acta Chem. Scand., 12 (1958) 2048. J. X. KHYM AND W. E. COHN, Biochim. Biophys. dcta, 15 (1954) 139. p. REICHARD, A. ]~ALDESTEN AND L. I~UTBERG, J. Biol. Chem., 236 (1961) 115o. p. REICHARD, J. Biol. Chem., 237 (1962) 3513. S. S. COHEN, H. D. BARNER AND J. LICHTENSTEIN, J. Biol. Chem., 236 (1961) 1448. H, G. KLEMPERER, J. S. KRAKOW A.ND E. S. CANELLAKIS, Biochim. Biophys. Acta, 61 (1962) 43. K. I~ANDERATH,Biochim. Biophys. Acta, 61 (1962) 852. it K. t{.ANDERATH, Thin-layer Chromatography, Verlag C h e m i e VVeinheim a n d e r B e r g s t r a s s e , a n d A c a d e m i c Press, N e w York, 1963 . 10 j . X. KHYM, L. P. ZILL AND W. E. COHN,in C. CALMON AND T. R. E. I'{RESSMAN, Ion-Exchangers in Organic and Biochemistry, Interscience, N e w York, 1957, p. 392. 11 W. E. COHN AND V. J. ]3OLLUM, Biochim. Biophys. Acta, 48 (1961) 588. z L. JAENICKE AND I. VOLLBRECHTSHAUSEN,
z it 4 5 e '/ 8
Received July ISt, 1963 Biochim. Biophys. Acta, 76 (1963) 622-624 SC 7114
Froctionotion of D N A I. Countercurrent distribution of notive ond degraded D N A Heterogeneity of DNA molecules from a given tissue has been examined by a number of methods including ultracentrifuga] sedimentation analysis and helix-coil transition during thermal denaturation 1, and ion-exchange chromatography *. 1RNA has been fractionated by countercurrent distribution in a number of 2-phase systems according to base composition3, 4 and countercurrent distribution has also been applied to the fractionation of soluble RNA's 5. Preliminary studies e with countercurrent distribution of degraded commercial I)NA from calf thymus achieved fractionation dependent upon base composition but undenatured I)NA remained largely in the aqueous phase and was not fractionated by these methods. In the present studies it has been possible to fractionate DNA's from various sources by countercurrent distribution. I)NA was prepared from rat liver, Drosophila melanogaster and Escherichia coli by modifications of the 4-aminosalicylate-phenol method 7. Commercial I)NA from salmon sperm (Mann, "highly polymerized") was reprecipitated from aqueous solution with ethoxyethanol in the presence of 4 % lithium acetate. Degradation was achieved enzymically in some experiments by treating a 0.2 % solution of DNA Biochim. Biophys. Acta, 76 (1963) 624 627
625
SHORT COMMUNICATIONS
containing o.ooi M magnesium acetate with IO mg DNAase (EC 3.1.4.5) per ml for 60 min at 20 ° and in other experiments by heating for 4 ° min at IOO° a o.I % solution of DNA containing o.ooi M sodium acetate and IO % phenol. The degraded DNA was precipitated with ethanol in the presence of 4 % lithium acetate. DNA degraded by heating in this way was found to have an S2o of 3.5-4.0 S as determined in the Spinco model E ultracentrifuge. Solvent systems for countercurrent distribution were similar to those developed for RNA 4 except that lithium citrate was added in place of potassium citrate in the aqueous phase, lithium salts being found to give more satisfactory partition of I)NA than potassium salts. The proportions in the countercurrent distribution system best suited to I)NA separation were: organic mixture, 28 volumes; amine solution, 12 volumes; water, 39.5 volumes; 0.033 M lithium citrate, 12.5 volumes. This system was designated as solvent system 15o/12.5. The lithium ion concentration was critical since in a 15o/13.o system the majority of the degraded I)NA remained in the first 20 tubes during 80 transfers while in a 15o/12.o system the majority travelled with the organic phase. Native I)NA showed a similar dependence on the lithium ion concentration though less marked in degree. 5-15 mg degraded I)NA or 1-'2 mg native ])NA were distributed in this solvent system (2 ml each phase) over 80 transfers. The I)NA was first dissolved in 2 ml of each phase of a 15012 system and before transferring the I)NA into the lower phase of the 15o/12.5 solvent system it was passed through a gradient of increasing concentration of lithium ion in single tube steps of solvent systems 15o/6, 15o/8, 15O/lO. This permitted optimal solubilization of I)NA in the organic phase (15o/12.5 system). After 80 transfers ethanol (I ml) was added to the contents of each tube to make one phase and the absorption read at 260 m F. For determination of base composition of denatured DNA fractions, 15 mg were used in countercurrent distribution and the contents of each lO-2O tubes pooled to give a small number of fractions. The DNA was recovered4 and base ratios s determined as described previously. Fig. I shows the patterns of distribution over 80 transfers of heat-degraded I)NA
.2•0
A260 1.5
!.i 1.C
•"•..: I
"'.••
•..:"-
0.~
2o 3'o
40
50
6O
70
8O
TPonsfer number
Fig. I. C o u n t e r c u r r e n t distribution p a t t e r n s of heat-degraded D N A ' s over 80 transfers in solvent s y s t e m I5O[I2. 5. D N A from: r a t liver ( - - - ) , s a l m o n s p e r m ( . . . ) , and E. coli ( ).
Biochim. Biophys. Acta, 76 (1963) 624-627
626
SHORT COMMUNICATIONS
from rat liver, salmon sperm and E. coli. A degree of coincidence exists between some peaks for DNA from rat liver and salmon sperm but in general marked differences exist among the 3 species illustrated. Reproducibility of pattern was good for DNA from the same tissue and species under the given conditions of degradation. As summarized in Table I this distribution shows trends in base composition similar to TABLE BASE
COMPOSITIONS
OF D E G R A D E D
DNA
BY COUNTERCURRENT
I FROM
RAT LIVER
FRACTIONATED
DISTRIBUTION
F r a c t i o n s are n u m b e r e d from the a q u e o u s end of the c o u n t e r c u r r e n t distribution. Heat-degraded D N A was fractionate~t in solvent s y s t e m i5o as described in the text, DNAase-degraded D N A in solvent s y s t e m i2 7 (see ref. 3). (Fractions I and 2 represented unfractionated D N A and have been omitted here.) Moles base/zoo moles Treatment
Fraction
Heat
I 2 3 4 3 4 5 6 7
DNAase
Guanine
Adenine
Cytosine
Thymine
24.9 26.0 21.5 16.3 29.4 27. 5 26.2 22.2 18.o
25.3 28.8 30.8 3 o.8 24. 9 26.2 29.4 32.1 32.9
24.9 20.8 18.7 20.5 22.2 20. 4 19.4 17. 7 19.1
24.9 24. 4 29.0 32.4 23. 5 25.9 25.0 28.0 3o.0
those found for RNA4 and for commercial DNA from calf thymus in other solvent systems e. DNA segments with a higher proportion of guanine and cytosine remained at the aqueous end while those with a higher proportion of adenine and thymine travelled further in the organic phase. Native DNA's (Fig. 2) showed a similar spread on countercurrent distribution in the I5O/I2. 5 solvent system but very different profiles from those of denatured
o.3t A26o
0.2
0.1 °
,
20
30 4Q 5Q £:K3 TFQRSfeP number
70
80
Fig. 2. C o u n t e r c u r r e n t distribution p a t t e r n s of native D N A ' s over 80 transfers in solvent s y s t e m I5O/I2. 5. D N A from: r a t liver ( - - - ) , D. melanogaster ( ), and E. coli (...).
Biochim. Biophys. Acta, 76 (1963) 624-6"27
SHORT COMMUNICATIONS
627
DNA's. For different preparations from the same tissue and species, reproducibility of profile was good with respect to general pattern but some quantitative variation occurred in individual peaks. This could be due to the small amount of native DNA which could be employed. Since insufficient material can be recovered from the countercurrent distribution to permit direct determination of base ratios of fractions, the basis of this fractionation of native DNA is not yet clear and other approaches to this problem are presently being studied. The fractionation of both native and degraded I)NA b y countercurrent distribution methods suggests considerable usefulness for this technique in the study of both base sequences and biological activities of DNA. We have found transforming activity to be retained b y native DNA from Bacillus subtilis after treatment with the 15o solvent system and this fact together with the degree of fractionation obtained with native DNA indicate the possible application of countercurrent distribution to separation of genetic functions of I)NA molecules. We wish to thank Mr. J. R. B. HASTINGS for the preparation of DNA from Drosophila melanogaster, Dr. M. GUEST for the preparation of DNA from Escherichia coli, Dr. A. D. Vlzoso for sedimentation measurements, and Professor A. HADDOW, F.R.S., for his interest in this work. This investigation has been supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research: Royal Cancer Hospital) from the Medical Research Council, the British Empire Cancer Campaign, the Anna Fuller Fund and the National Cancer Institute of the National Institutes of Health, U.S. Public Health Service. One of us (C.K.) is Arthur A. Thomas Cancer Research Fellow of the AntiCancer Council of Victoria, on leave from the Baker Medical Research Institute, Melbourne. Chester Beatty Research Institute, Institute o/ Cancer Research: Royal Cancer Hospital, F u l h a m Road, London (Great Britain)
CHEVKIDSON K. S. KIRBY
1 p. DOTY,J, MARMURAND 1~. SUEOKA,Broohhaven Syrup. Biol., 12 (1959) I. 2 H. S. I~.OSENKRANZANDA. BENDICH,J. Am. Chem. Soc., 81 (1959) 9o2. * K. S. KIRBY, Biochim. Biophys. Acta, 61 (1962) 5o6. 4 K. S. KIRBY,J. R. B. HASTINGSANDM. A. O'SULLIVAN,Biochim. Biophys. Acta, 61 (1962) 978. R. W. HOLLEY,J. APGARA.NDB. P. DOCTOR,Ann. N.Y. Acad. Sci., 88 (196o) 745. e K. S. KIRBY, The Molecular Basis o/Neoplasia, Texas Univ. Press, 1962, p. 59. K. S. KIRBY. Biochim. Biophys. Acta, 36 (1959) 117. s K. S. KIRBY, Biochem. J., 66 (1957) 495. Received J u l y 4th, 1963 Biochim. Biophys. Aaa, 76 (1963) 624-627