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Isolation of high-molecular-weight DNA from amphibian erythrocytes
BIOCHIMICAET BIOPHYSICAACTA
353
BBA 96859
I S O L A T I O N OF H I G H - M O L E C U L A R - W E I G H T DNA FROM A M P H I B I A N ERYTHROCYTES D. K. CHATTORAJ* Biophysics Laboratory, Saha Institute o] Nuclear Physics, Calcutta - 37 (India)
(Received December 4th, 197o)
SUMMARY Chromatin from nucleated erythrocytes of an amphibian, Bu]o mdanostictus, when dialysed against i M NaCl-o.oi M E D T A (pH 8.0) solution and spread b y the Kleinschmidt technique for electron microscopy, revealed randomly aggregated networks of very thin fibrils (comparable to that of protein-free DNA molecules). Free ends were rare. Further digestion with pronase (I mg/ml) eliminated aggregation and showed single, linear DNA molecules of varied lengths. If shear forces were avoided as far as possible, molecules up to a m a x i m u m of 78 # m long were obtained.
From chemical studies it is known that the DNA content per eukaryotic cell is about a thousand times larger than t h a t in a bacterium. I t is not known whether the entire DNA in each chromosome is present as a single unit of continuous 3',5'phosphodiester links or, alternatively, whether there are more ttmn one piece, which are either linked b y non-DNA material or free from each other. The failure to answer these questions lies in the extreme shear sensitivity of DNA which makes isolation of long DNA molecules without breakage extremely difficult. The conventional methods of DNA isolation from a variety of bacteria and eukaryotic cells seldom give molecules longer than about 30 million daltons, representing only about one hundredth of the bacterial genome, irrespective of the source 1. However, with the use of proteolytic enzymes for deproteinization and more careful manipulation of the DNA sample in other steps, shear forces could be substantially reduced and molecules much longer than reported before could be isolated from a variety of species (for review see ref. 2). The most commonly used method for estimation of length in these studies was either autoradiography or electron microscopy 2. Apart from determinations of length, the employment of visual techniques also throws light upon the question of circularity of eukaryotic DNA 3 as is often found in viruses, bacteria or mitochondria, or the presence of a n y disorganized region in the DNA molecule a. These studies were, however, too few to draw a n y generalized conclusion. The present study reports another attempt to isolate eukaryotic DNA avoiding shear as far as possible. Under the electron microscope only single, linear, unblemished, double-stranded molecules up to a m a x i m u m of about 7 8 / , m long were seen. * Present address: Biophysics Laboratory, The University of Wisconsin, 1525 Linden Drive, Madison, Wisc. 53706, U.S.A. Biochim. Biophys. Acta, 240 (1971) 353-357
354
D.K. CHATTORAJ
The washed erythrocytes from an amphibian, toad (Bu/o melanostictus), were hemolysed in o.o15 M NaCl-o.ooI 5 M sodium citrate in the cold (0-5°). The lysate was clarified by centrifugation and the pellet (gel) was put into a solution of I M NaCl-o.oI M E D T A in o.oi M Tris buffer (pH 8.0, adjusted with HC1) and dialysed against the same solution for 20 h in the cold. The gel was not completely dispersed. A part of the relatively clearer portion of the gel was mixed with an equal volume of 0.04 °/o cytochrome c (Sigma Chemicals) in I M ammonium acetate and spread onto the surface of o.I M ammonium acetate solution following the method of Kleinschmidt. To the other part, pronase (Calbiochem) was added to a final concentration of I mg/ml, and incubated at 60 ° for 4 h. The solution was rolled with chloroformisoamyl alcohol mixture (24 : I, v/v) at 22 rev./min for IO rain and centrifuged. The supernatant was digested with pancreatic deoxyribonuclease I in some cases as described previously 5. For electron microscopy, the supernatant was spread with cytochrome c as above. The splead film was sampled for electron microscopy as described earlier ~. Electron micrographs were taken under the Siemens Elmiskop I operated at 60 kV. The errors due to variation in magnification of the microscope and in the measurement of length of DNA molecules were found to be within 7 %. When the nucleoprotein gel dialysed against saline-EDTA solution was spread, long and very thin fibrils always interconnecting each other into highly aggregated networks were seen (Fig. I). They rarely showed free ends. The diameter of the unit fibril was very uniform and could be followed for lengths up to IO #m, after which it was lost in the aggregated structures. Often two or more fibrils were aggregated along their length over several micrometers otherwise the aggregation appeared to be random. Aggregation at this stage, where deproteinization was hardly complete, could represent the gelling of the nucleoprotein molecules 6 and as such might have no bearing on the network of interphase chromatin 2,~. Nevertheless, it is clearly demonstrated that saline-EDTA can disrupt the structure of 25o-• chromatin fibers e, 5 and nearly reduce them to the dimension of protein-free DNA molecules. It should be mentioned that the fiblils in Fig. I were also in contact with ammonium acetate during spreading for electron microscopy. Since ammonium acetate by itself can dissociate nucleohistones 7, the observed changes might be a combined effect of both saline-EDTA and ammonium acetate. However, the total time involved in spreading by the Kleinschmidt technique was less than IO rain whereas the dialysis time was 20 h. When the above sample was spread after treatment with pronase, aggregation was found to be completely absent. The continuity of the fibrils were destroyed by deoxyribonuclease within a few minutes. When spread at a lower concentration, the lengths of the DNA molecules could be followed from end to end (Fig. 2) and varied from less than I/~iil to more than 78/~m (Fig. 3)- The variable lengths of the molecules and preponderance of shorter molecules indicate that degradation has taken place, and as such the m a x i m u m length obtained (78/~m) merely gives an idea of the lower limit rather than the actual length of the molecules in vivo. Aside from length, other conformational characteristics of these molecules were very similar to those isolated from the same specimen b y a modification a of the method of MARMUR 13.
Short pieces of single-stranded tails were sometimes found attached to D N A but they were much shorter than the length reported for Chironomus salivary gland Biochim. Biophys, Acta, 240 (1971) 353-357
HIGH-MOLECULAR-WEIGHT
DNA FROM ERYTHROCYTES
355
Fig, I. C h r o m a t i n from toad, dialysed a g a i n s t s a l i n e - E D T A sOlution a n d s p r e a d b y t h e I~leins c h m i d t t e c h n i q u e . Circularly s h a d o w e d with P t - I r (IO %) vapor. H i g h l y a g g r e g a t e d n e t w o r k of v e r y t h i n fibrils c a n be seen. Magnification, x 29 600. Fig, 2. S a m e as Fig. i, b u t f u r t h e r t r e a t e d w i t h p r o n a s e after dialysis. T h e figure s h o w s a molecule a b o u t 7 ° H m long. Magnification, × 29 600.
356
D.K. CHATTORAJ ]
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Length (p.rn) Fig. 3. Length distribution of the long DNA molecules obtained by electron microscopy. Many of the long molecules could not be measured because of the ambiguity associated with highly looped structures and because long molecules ran out of the field of view. Because of these complications long molecules were underestimated; additionally, many molecules less than 25/~m were ignored. DNA filaments*. In the latter case, the single-stranded tails were interpreted as points of replication and/or transcription*. Since single-stranded tails were rarely seen in the present study it appears that they were produced during shear breakage. I t should also be mentioned that erythrocyte DNA does not replicate or transcribe. Apart from single-stranded tails no other structural abnormalities were seen. The a t t e m p t to isolate DNA molecules without shear in the present study was similar to the method of HOTTA AND BASSEL3 where the authors reported a part of the DNA molecules from boar sperm to be in circular form. Though the m a x i m u m length surviving in the present isolation method was much more than that (31 #m) reported 3, circular DNA was never found. Using crude cell lysate, still longer stretches of the molecule were seen (lO8/~m, sea urchin spermS; 154.5/~m Chironomus thummi*), but always in the linear form. Thus, circularity of eukaryotic DNA in the order of the length reported 3 is unfounded in all the later studies. Moreover, the origin of the circular DNA in boar sperm might be mitochondrial 2. Since visual techniques have permitted tracing of very long DNA molecules (832/~m, Haemophilus in]luenzaeg; I8OO/~m, Chinese hamster1°; 15o jura Spinach chloroplasts11; 193 # m replicating T412) in crude preparations, it seems that test tube isolation of a relatively protein-free (maximum protein contamination in the present study was 3 %) DNA sample of any longer than about 50/zm is extremely difficult at the present stage. I thank Prof. N. N. Das Gupta for encouragement and support. I am grateful to Dr. Ross B. Inman, the University of Wisconsin, for improving the manuscript and to Dr. D. N. Misra for helping with the electron microscope. Financial assistance was received from the Department of Atomic Energy, Government of India.
REFERENCES I D. K. CHATTORAJ, J. CHAKRABORTY AND P. SADHUKItAN, J. Electronmicroscopy Tokyo, i8 (1969) 272. 2 H. R I s AND D. F. KUBAI, Ann. Rev. Genet., 4 (197 ° ) 263. 3 Y. HOTTA AND A. BASSEL, Proc. Natl. Acad. Sci. U.S., 53 (1965) 356. 4 ~). R. WOLST]~NHOLME, I. B. DAWlI) AND H. J. RmTOW, J. Cell Biol., 35 (1967) I45A5 D. I~. CHATTORAJ, ]:). SADHUKHAN AND J. CHAKRABORTY, Exptl. Cell Res., 53 (1968) 65. 6 G. ZUB,tY AND P. DOTY, J. Mol. Biol., I (1959) I.
Biochim. Biophys. Acta, 240 (1971) 353-357
HIGH-MOLECULAR-WEIGHT D N A I~ROM ERYTHROCYTES 7 8 9 IO II I2 13
357
D. BASTIA AND M. S. SWAMINATHAN,Exptl. Cell Res., 48 (I967) i8. A. J. SOLA~I, J. Ultrastruct. Res., 17 (I967) 42I. L. A. MAcHATTIE, K. I. BERNS AND C. A. THOMAS, J. Mol. Biol., 11 (I965) 648. J. A. HUBERMAN A~D A. D. RIGGS, Proc. Natl. Acad. Sci. U.S., 55 (I966) 599. C. L. F. WOODCOCK AND H. FER,~NNDEZ-IVfORMq,J. Mol. Biol., 31 (I968) 627. J. A. HUBERMA~;, Cold spring Harbor Symp. Quant. Biol., 33 (I968) 5o9. J. I~ARMUR, J. Mol. Biol., 3 (I97 I) 2o8.
Biochim. Biophys. Acla, 24o (I97 I) 353-359
353
BBA 96859
I S O L A T I O N OF H I G H - M O L E C U L A R - W E I G H T DNA FROM A M P H I B I A N ERYTHROCYTES D. K. CHATTORAJ* Biophysics Laboratory, Saha Institute o] Nuclear Physics, Calcutta - 37 (India)
(Received December 4th, 197o)
SUMMARY Chromatin from nucleated erythrocytes of an amphibian, Bu]o mdanostictus, when dialysed against i M NaCl-o.oi M E D T A (pH 8.0) solution and spread b y the Kleinschmidt technique for electron microscopy, revealed randomly aggregated networks of very thin fibrils (comparable to that of protein-free DNA molecules). Free ends were rare. Further digestion with pronase (I mg/ml) eliminated aggregation and showed single, linear DNA molecules of varied lengths. If shear forces were avoided as far as possible, molecules up to a m a x i m u m of 78 # m long were obtained.
From chemical studies it is known that the DNA content per eukaryotic cell is about a thousand times larger than t h a t in a bacterium. I t is not known whether the entire DNA in each chromosome is present as a single unit of continuous 3',5'phosphodiester links or, alternatively, whether there are more ttmn one piece, which are either linked b y non-DNA material or free from each other. The failure to answer these questions lies in the extreme shear sensitivity of DNA which makes isolation of long DNA molecules without breakage extremely difficult. The conventional methods of DNA isolation from a variety of bacteria and eukaryotic cells seldom give molecules longer than about 30 million daltons, representing only about one hundredth of the bacterial genome, irrespective of the source 1. However, with the use of proteolytic enzymes for deproteinization and more careful manipulation of the DNA sample in other steps, shear forces could be substantially reduced and molecules much longer than reported before could be isolated from a variety of species (for review see ref. 2). The most commonly used method for estimation of length in these studies was either autoradiography or electron microscopy 2. Apart from determinations of length, the employment of visual techniques also throws light upon the question of circularity of eukaryotic DNA 3 as is often found in viruses, bacteria or mitochondria, or the presence of a n y disorganized region in the DNA molecule a. These studies were, however, too few to draw a n y generalized conclusion. The present study reports another attempt to isolate eukaryotic DNA avoiding shear as far as possible. Under the electron microscope only single, linear, unblemished, double-stranded molecules up to a m a x i m u m of about 7 8 / , m long were seen. * Present address: Biophysics Laboratory, The University of Wisconsin, 1525 Linden Drive, Madison, Wisc. 53706, U.S.A. Biochim. Biophys. Acta, 240 (1971) 353-357
354
D.K. CHATTORAJ
The washed erythrocytes from an amphibian, toad (Bu/o melanostictus), were hemolysed in o.o15 M NaCl-o.ooI 5 M sodium citrate in the cold (0-5°). The lysate was clarified by centrifugation and the pellet (gel) was put into a solution of I M NaCl-o.oI M E D T A in o.oi M Tris buffer (pH 8.0, adjusted with HC1) and dialysed against the same solution for 20 h in the cold. The gel was not completely dispersed. A part of the relatively clearer portion of the gel was mixed with an equal volume of 0.04 °/o cytochrome c (Sigma Chemicals) in I M ammonium acetate and spread onto the surface of o.I M ammonium acetate solution following the method of Kleinschmidt. To the other part, pronase (Calbiochem) was added to a final concentration of I mg/ml, and incubated at 60 ° for 4 h. The solution was rolled with chloroformisoamyl alcohol mixture (24 : I, v/v) at 22 rev./min for IO rain and centrifuged. The supernatant was digested with pancreatic deoxyribonuclease I in some cases as described previously 5. For electron microscopy, the supernatant was spread with cytochrome c as above. The splead film was sampled for electron microscopy as described earlier ~. Electron micrographs were taken under the Siemens Elmiskop I operated at 60 kV. The errors due to variation in magnification of the microscope and in the measurement of length of DNA molecules were found to be within 7 %. When the nucleoprotein gel dialysed against saline-EDTA solution was spread, long and very thin fibrils always interconnecting each other into highly aggregated networks were seen (Fig. I). They rarely showed free ends. The diameter of the unit fibril was very uniform and could be followed for lengths up to IO #m, after which it was lost in the aggregated structures. Often two or more fibrils were aggregated along their length over several micrometers otherwise the aggregation appeared to be random. Aggregation at this stage, where deproteinization was hardly complete, could represent the gelling of the nucleoprotein molecules 6 and as such might have no bearing on the network of interphase chromatin 2,~. Nevertheless, it is clearly demonstrated that saline-EDTA can disrupt the structure of 25o-• chromatin fibers e, 5 and nearly reduce them to the dimension of protein-free DNA molecules. It should be mentioned that the fiblils in Fig. I were also in contact with ammonium acetate during spreading for electron microscopy. Since ammonium acetate by itself can dissociate nucleohistones 7, the observed changes might be a combined effect of both saline-EDTA and ammonium acetate. However, the total time involved in spreading by the Kleinschmidt technique was less than IO rain whereas the dialysis time was 20 h. When the above sample was spread after treatment with pronase, aggregation was found to be completely absent. The continuity of the fibrils were destroyed by deoxyribonuclease within a few minutes. When spread at a lower concentration, the lengths of the DNA molecules could be followed from end to end (Fig. 2) and varied from less than I/~iil to more than 78/~m (Fig. 3)- The variable lengths of the molecules and preponderance of shorter molecules indicate that degradation has taken place, and as such the m a x i m u m length obtained (78/~m) merely gives an idea of the lower limit rather than the actual length of the molecules in vivo. Aside from length, other conformational characteristics of these molecules were very similar to those isolated from the same specimen b y a modification a of the method of MARMUR 13.
Short pieces of single-stranded tails were sometimes found attached to D N A but they were much shorter than the length reported for Chironomus salivary gland Biochim. Biophys, Acta, 240 (1971) 353-357
HIGH-MOLECULAR-WEIGHT
DNA FROM ERYTHROCYTES
355
Fig, I. C h r o m a t i n from toad, dialysed a g a i n s t s a l i n e - E D T A sOlution a n d s p r e a d b y t h e I~leins c h m i d t t e c h n i q u e . Circularly s h a d o w e d with P t - I r (IO %) vapor. H i g h l y a g g r e g a t e d n e t w o r k of v e r y t h i n fibrils c a n be seen. Magnification, x 29 600. Fig, 2. S a m e as Fig. i, b u t f u r t h e r t r e a t e d w i t h p r o n a s e after dialysis. T h e figure s h o w s a molecule a b o u t 7 ° H m long. Magnification, × 29 600.
356
D.K. CHATTORAJ ]
1
I0
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20
30
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40
50
60
70
80
Length (p.rn) Fig. 3. Length distribution of the long DNA molecules obtained by electron microscopy. Many of the long molecules could not be measured because of the ambiguity associated with highly looped structures and because long molecules ran out of the field of view. Because of these complications long molecules were underestimated; additionally, many molecules less than 25/~m were ignored. DNA filaments*. In the latter case, the single-stranded tails were interpreted as points of replication and/or transcription*. Since single-stranded tails were rarely seen in the present study it appears that they were produced during shear breakage. I t should also be mentioned that erythrocyte DNA does not replicate or transcribe. Apart from single-stranded tails no other structural abnormalities were seen. The a t t e m p t to isolate DNA molecules without shear in the present study was similar to the method of HOTTA AND BASSEL3 where the authors reported a part of the DNA molecules from boar sperm to be in circular form. Though the m a x i m u m length surviving in the present isolation method was much more than that (31 #m) reported 3, circular DNA was never found. Using crude cell lysate, still longer stretches of the molecule were seen (lO8/~m, sea urchin spermS; 154.5/~m Chironomus thummi*), but always in the linear form. Thus, circularity of eukaryotic DNA in the order of the length reported 3 is unfounded in all the later studies. Moreover, the origin of the circular DNA in boar sperm might be mitochondrial 2. Since visual techniques have permitted tracing of very long DNA molecules (832/~m, Haemophilus in]luenzaeg; I8OO/~m, Chinese hamster1°; 15o jura Spinach chloroplasts11; 193 # m replicating T412) in crude preparations, it seems that test tube isolation of a relatively protein-free (maximum protein contamination in the present study was 3 %) DNA sample of any longer than about 50/zm is extremely difficult at the present stage. I thank Prof. N. N. Das Gupta for encouragement and support. I am grateful to Dr. Ross B. Inman, the University of Wisconsin, for improving the manuscript and to Dr. D. N. Misra for helping with the electron microscope. Financial assistance was received from the Department of Atomic Energy, Government of India.
REFERENCES I D. K. CHATTORAJ, J. CHAKRABORTY AND P. SADHUKItAN, J. Electronmicroscopy Tokyo, i8 (1969) 272. 2 H. R I s AND D. F. KUBAI, Ann. Rev. Genet., 4 (197 ° ) 263. 3 Y. HOTTA AND A. BASSEL, Proc. Natl. Acad. Sci. U.S., 53 (1965) 356. 4 ~). R. WOLST]~NHOLME, I. B. DAWlI) AND H. J. RmTOW, J. Cell Biol., 35 (1967) I45A5 D. I~. CHATTORAJ, ]:). SADHUKHAN AND J. CHAKRABORTY, Exptl. Cell Res., 53 (1968) 65. 6 G. ZUB,tY AND P. DOTY, J. Mol. Biol., I (1959) I.
Biochim. Biophys. Acta, 240 (1971) 353-357
HIGH-MOLECULAR-WEIGHT D N A I~ROM ERYTHROCYTES 7 8 9 IO II I2 13
357
D. BASTIA AND M. S. SWAMINATHAN,Exptl. Cell Res., 48 (I967) i8. A. J. SOLA~I, J. Ultrastruct. Res., 17 (I967) 42I. L. A. MAcHATTIE, K. I. BERNS AND C. A. THOMAS, J. Mol. Biol., 11 (I965) 648. J. A. HUBERMAN A~D A. D. RIGGS, Proc. Natl. Acad. Sci. U.S., 55 (I966) 599. C. L. F. WOODCOCK AND H. FER,~NNDEZ-IVfORMq,J. Mol. Biol., 31 (I968) 627. J. A. HUBERMA~;, Cold spring Harbor Symp. Quant. Biol., 33 (I968) 5o9. J. I~ARMUR, J. Mol. Biol., 3 (I97 I) 2o8.
Biochim. Biophys. Acla, 24o (I97 I) 353-359