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Purification of the desoxypentose nucleic acid of Hemophilus influenzae having transforming activity
Purification of the Desoxypentose Nucleic Acid of Hemophilus influenzae Having Transforming Activity’ Stephep Zamenhof, Grace Leidy, Hattie E. Alexander, Patricia L. FitzGerald and Erwin Chargaff From
the Departments
of Biochemistry
Surgeons,ColumbiaUniversity, and
and Pediatrics, College The Babies Hospital, New
Received April
of Physicians York,
New
and
York
22, 1952
INTRODUCTION
The important discovery by Avery and collaborators (2) that certain microbial desoxypentose nucleic acid (DNA) preparations are endowed with transforming activity has made it of interest to develop methods for the purification and chemical characterization of agents of this type. The finding that active transforming preparations consist almost entirely of highly polymerized DNA does not in itself prove the exclusive function of the latter, nor does the destruction of biological activity by enzymes presumed to act specifically on DNA (2, 3, 4) demonstrate more than that DNA is an indispensable participant in the transforming phenomenon. Regardless of what the final explanation of the mechanisms of microbial transformations will be, it is of importance to follow the changes in transforming activity attending the removal of substances other than highly polymerized DNA from active preparations. The present paper deals with the purification of transforming preparations from Hernophilus injtuenzae (4). Crude solutions of bacterial-transforming principles contain, as ‘the main nondialyzable components, DNA’s, pentose nucleic acids (PNA’s), proteins, and polysaccharides. The removal of at least the bulk of the proteins without the destruction of DNA presents no difficulties when the procedure of Sevag (5) is employed (2). The extent to which the * This work has been supported by research grants from the National Institutes of Health, U. S. Public Health Service, and from the Rockefeller Foundation, Part of it w&s presented at the XIIth International Congress of Pure and Applied Chemistry, New York, September 10-13, 1951 (1). 50
DNA OF IX. INFLUENZAE
51
presence of protein impurities can be excluded will depend not only on the sensitivity of the test, but also on the composition and properties of the particular proteins. Polysaccharides have been eliminated in a few instances (2, 4, 6), use being made of enzymes, where available, specifically destroying certain bacterial polysaccharides, or of fractionation procedures of limited applicability. For the latter reason, it has not yet been possible to purify the transforming principles of several bacterial types. The extent to which PNA had been removed from previously described transforming preparations does not appear to have been recorded. The use of crystalline ribonuclease, to digest this ubiquitous contaminant, has been suggested(6). This expedient is, however, not devoid of dangers; the enzyme preparations sometimes contain traces of pancreatic desoxyribonuclease; the enzymatic degradation of PNA to diffusible fragments is not complete [cf. (7-lo)]. The method of purification presented here is based on the observation that -the electrophoretic mobility of DN,4 exceeds that of PNA (11, 12). This difference, sufficient for separation, applies even to DNA and PNA from the same source (12). Moreover, owing to the very high electrophoretic mobility of DNA this separation procedure will eliminate not only the much more slowly moving immunologically active polysaccharides, but also many other impurities that may be present.2 Tb.e composition of one of the DNA preparations isolated in this manner is discussedin the forthcoming publication (13). EXPERIMENTAL
Analytical Procedures Micro modifications of existing methods served for the analyses on intact prepsrations; they will on occasion be presented in detail. The DNA content was estimated calorimetrically by the reaction with diphenylamine (14), the PNA content by means of the reactions with orcinol (15) or cysteine (16), and the protein content by an adaptation of the biuret reaction. The immunologically active polysaccharides were assayed by serological methods (4). The estimation of transforming activity was carried out as described previously (4).
Preparation and Purification Preparation of Crude Extracts. Hemophilus infEuenzae, type b or type c, W&B grown on Levinthal agar plates, harvested, and lysed with sodium desoxycholate * It should be emphasized that the electrophoretic purification procedure as such does not bring about the geparation of proteins remaining in the solution from the DNA fraction.
52
ZAMENHOF,
LEIDY,
ALEXANDER,
FITZGERALD
AND
CHARGAFF
solution; the extracts were partly freed of protein by treatment with a chloroformpentand mixture, and the crude transforming preparations were precipitated with ethanol, essentially as described before (4). The separated fibers or the entire precipitates were dissolved in 0.15 M aqueous sodium chloride, which was 0.02 M in respect to sodium phosphate or sodium citrate of pH 7.2-7.4, and the solution was dialyzed overnight in the cold against the 200-fold volume of the same solvent. The processing of the entire precipitate was more advantageous in regard to the final DNA yield than when the threads alone were used. The solutions contained l-3.3 mg. DNA/ml. This fractionation stage is described in Table I as step No. 1. PuriJication. The separation of DNA from PNA. polysaccharides, etc., was performed at 1.5” in a Tiselius cell.8 When a crude solution in the NaCl-phosphate buffer mentioned above was subjected to electrophoresis, the ascending mobility of DNA was found as -17 X lo+, that of PNA as -12.3 X KV sq. cm./v./sec., a difference in itself not adequate for the separation of sufficient amounts of DNA TABLE Purification
of Transforming
I
Preparations Types b and
from
Hemophilus
influensae,
c steps No. 1
PNA, % of DNA Protein, % of DNA Immunologically active DNA Lowest concentration ducing transformation type typr
b c
polysaccharides, of DNA
(pg./ml.)
‘% of
160-164 21-33 >30
No. 2
No. 3
<1 C2.5 <0.3
in<0.062
<0.0005
<0.0004
in the course of its rapid passage through the cell. Automatic slow compensation was, therefore, applied in order to prolong the electrophoretic separation up to 67 hr., at an electrode potential of 120 v. Owing to the high molecular weight and the high viscosity of DNA, this could be done without undue disturbance of the boundaries; even at the end of this period the DNA boundary presented itself, in fact, as a single hypersharp peak (see Fig. 1). The PNA peak flattened during this time because of diffusion, but was still sufficiently distinct for clean separation. When the DNA and PNA peaks, constantly visible owing to the compensstion, had separated by 2(r25 mm., the compensation was stopped and the DNA boundary permitted to migrate into the clean top section of the cell,’ until the 3 The instrument used was model 38 of the Perkin-Elmer Corporation, Glenbrook, Conn. Cells of a capacity of 2 or 6 ml. were employed; a special extension tube in the top section of the cell permitted the separation of larger volumes. We are grateful to Dr. D. H. Moore of this College for help and advice. 4 This step is necessary because the lower sections of the cell are contaminated by the crude viscous solution.
DNA
OF H. INFLJJENZAE
53
foot of the PNA peak nearly touched the dividing line between the cell sections. Following the separation of the sections in the usual way, the DNA fraction was collected from the top section. Up to 25% of the DNA contained in the original solution was thus obtained. The remainder was recovered from the other cell sections and resubjected to electrophoresis two to four more times. When it became too diluted, its cont,ents were precipitated with 2 vol. ethanol, dialyzed, and subjected to electrophoretic separation as before, though in a smaller cell. The combined DNA fractions from two to four electrophoresis runs (step No. 2 in Table I) were precipit,ated with 2 vol. ethanol; the resulting threads were
FIG. 1. Electrophoresis pattern (ascending) of crude transforming preparation from Hemophilus in$uenzae, type c, containing 1.82 mg. DNA/ml.; after 336 min. at 6.3 V./cm. in citrate-chloride buffer of pH 7.25, ionic strength 0.2. The faster sharp peak on the left side belongs to DNA, that on the right side to PNA. lifted and dissolved in l-2 ml. of 10% aqueous sodium chloride. For the stepwise removal of protein, the solution was shaken four times, each for 12 hr., with g vol. chloroform%-pentanol (3:l). The DNA, recovered finally in fibrous form, by the addition of 2 vol. ethanol, was dissolved in l-2 ml. of 0.16 M sodium chloride solution, to yield a clear viscous liquid. The final preparations are listed in Table I as step No. 3. All operations were carried out in the cold. RESULTS is illustrated
The progress of purification in Table I. Unless specified, ‘Ihe data refer to preparations of the transforming principles from both
54
ZAMENHOF,
LEIDY,
ALEXANDER,
FITZGERALD
AND
CHARGAFF
types b and c. It will be seen that the electrophoretic separation of DNA eliminated practically all PNA and the immunologically active polysaccharides. With respect to the latter, the limits of sensitivity of the serological reactions must, of course, be taken into account and the estimates are by necessity of no great accuracy. The final preparations were active in inducing transformations in concentrations of less than 0.0004 pg./ml. (type b) and less than 0.01 pg./ml. (type c). When the solution of the final DNA preparation of type c was dialyzed, lyophilized, and dried in 2racu.oat 60” over PaOaoperations destructive of transforming activity-the residue assayed for 96% DNA and contained N 15.75 and P 8.850/,. These results as well as those on the purine and pyrimidine composition of this preparaCon, submitted in the forthcoming paper (13), exclude the presence of major impurities in the DNA. No decrease in the transforming activity per weight unit of DNA was noted with the preceding purification and removal of proteins and other admixtures. In the case of the preparation from type b the electrophoretic separation in particular seemed, in fact, to yield a more active DNA. The results strongly suggest the importance of the DNA of Hemophdua influenzae or of a fraction contained in it for the transformation reaction under discussion. SUMMARY
The purification of DNA preparations of Hemophilus injluenzae, types b and c, endowed with transforming activity is described. The mild procedure makes use of electrophoretic separation of the DNA from contaminating PNA and polysaccharides. The final DNA preparations, free of detectable amounts of these contaminants and of proteins, were active in transformation in concentrations of less than 0.0004 pg. and 0.01 pg. DNA/ml. for types b and c, respectively. REFERENCES 1. ZAMENHOF, S., LEIDY, G., ALEXANDER. H. E., FITZGERALD, P. L., AND CEIARGAFF, E., Abstracts, p. 100. XIIth International Congress of Pure and Applied Chemistry, New York, September 1951. 2. AVERY, 0. T., MACLEOD, C. M., AND MCCARTY, M., J. Exptl. Med. 79, 137 (1944). 3. MCCARTY, M., AND AVERY, 0. T., J. Exptl. Med. 83,89 (1946). 4. ALEXANDER, H. E., AND LEIDY, G., Proc. Sot. Exptl. Biol. Med. 73,435 (1950); J. Ezptl. Med. 93, 345 (1951).
DNA
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
OF
H.
INFLUENZAE
55
SEVAG, M. G., Biochem. 2.273, 419 (1934). MCCARTY, M., AND AVERY, 0. T., J. Ezptl. Med. 83,97 (1946). KUNITZ, M., J. Gen. Physiol. 24, 15 (1940). LORING, H. S., CARPENTER, F. H., AND ROLL, P. M., J. Biol. Chem. 169, 601 (1947). SCHMIDT, G., CUBILES, R., SWARTZ, B. H., AND THANNHAUSER, S. J., J. Biol. Chem. 170, 759 (1947). MA.GASANIK, B., AND CHARGA~F, E., Biochem. et Biophys. Acta 7, 396 (1951). COHEN, S. S., J. Biol. Chem. 146, 471 (1942). CHARGAFF, E., AND ZAMENHOF, S., J. Biol. Chem. 173, 327 (1948). ZAMENHOF, S., BRAWERMAN, G., AND CHARGAFF, E., in preparation. DISCHE, Z., Mikrochemie 8, 4 (1930). MIUBAUM, W., 2. physiol. Chem. 208, 117 (1939). DIYCHE, Z., J. Biol. Chem. 181, 379 (1949).
the Departments
of Biochemistry
Surgeons,ColumbiaUniversity, and
and Pediatrics, College The Babies Hospital, New
Received April
of Physicians York,
New
and
York
22, 1952
INTRODUCTION
The important discovery by Avery and collaborators (2) that certain microbial desoxypentose nucleic acid (DNA) preparations are endowed with transforming activity has made it of interest to develop methods for the purification and chemical characterization of agents of this type. The finding that active transforming preparations consist almost entirely of highly polymerized DNA does not in itself prove the exclusive function of the latter, nor does the destruction of biological activity by enzymes presumed to act specifically on DNA (2, 3, 4) demonstrate more than that DNA is an indispensable participant in the transforming phenomenon. Regardless of what the final explanation of the mechanisms of microbial transformations will be, it is of importance to follow the changes in transforming activity attending the removal of substances other than highly polymerized DNA from active preparations. The present paper deals with the purification of transforming preparations from Hernophilus injtuenzae (4). Crude solutions of bacterial-transforming principles contain, as ‘the main nondialyzable components, DNA’s, pentose nucleic acids (PNA’s), proteins, and polysaccharides. The removal of at least the bulk of the proteins without the destruction of DNA presents no difficulties when the procedure of Sevag (5) is employed (2). The extent to which the * This work has been supported by research grants from the National Institutes of Health, U. S. Public Health Service, and from the Rockefeller Foundation, Part of it w&s presented at the XIIth International Congress of Pure and Applied Chemistry, New York, September 10-13, 1951 (1). 50
DNA OF IX. INFLUENZAE
51
presence of protein impurities can be excluded will depend not only on the sensitivity of the test, but also on the composition and properties of the particular proteins. Polysaccharides have been eliminated in a few instances (2, 4, 6), use being made of enzymes, where available, specifically destroying certain bacterial polysaccharides, or of fractionation procedures of limited applicability. For the latter reason, it has not yet been possible to purify the transforming principles of several bacterial types. The extent to which PNA had been removed from previously described transforming preparations does not appear to have been recorded. The use of crystalline ribonuclease, to digest this ubiquitous contaminant, has been suggested(6). This expedient is, however, not devoid of dangers; the enzyme preparations sometimes contain traces of pancreatic desoxyribonuclease; the enzymatic degradation of PNA to diffusible fragments is not complete [cf. (7-lo)]. The method of purification presented here is based on the observation that -the electrophoretic mobility of DN,4 exceeds that of PNA (11, 12). This difference, sufficient for separation, applies even to DNA and PNA from the same source (12). Moreover, owing to the very high electrophoretic mobility of DNA this separation procedure will eliminate not only the much more slowly moving immunologically active polysaccharides, but also many other impurities that may be present.2 Tb.e composition of one of the DNA preparations isolated in this manner is discussedin the forthcoming publication (13). EXPERIMENTAL
Analytical Procedures Micro modifications of existing methods served for the analyses on intact prepsrations; they will on occasion be presented in detail. The DNA content was estimated calorimetrically by the reaction with diphenylamine (14), the PNA content by means of the reactions with orcinol (15) or cysteine (16), and the protein content by an adaptation of the biuret reaction. The immunologically active polysaccharides were assayed by serological methods (4). The estimation of transforming activity was carried out as described previously (4).
Preparation and Purification Preparation of Crude Extracts. Hemophilus infEuenzae, type b or type c, W&B grown on Levinthal agar plates, harvested, and lysed with sodium desoxycholate * It should be emphasized that the electrophoretic purification procedure as such does not bring about the geparation of proteins remaining in the solution from the DNA fraction.
52
ZAMENHOF,
LEIDY,
ALEXANDER,
FITZGERALD
AND
CHARGAFF
solution; the extracts were partly freed of protein by treatment with a chloroformpentand mixture, and the crude transforming preparations were precipitated with ethanol, essentially as described before (4). The separated fibers or the entire precipitates were dissolved in 0.15 M aqueous sodium chloride, which was 0.02 M in respect to sodium phosphate or sodium citrate of pH 7.2-7.4, and the solution was dialyzed overnight in the cold against the 200-fold volume of the same solvent. The processing of the entire precipitate was more advantageous in regard to the final DNA yield than when the threads alone were used. The solutions contained l-3.3 mg. DNA/ml. This fractionation stage is described in Table I as step No. 1. PuriJication. The separation of DNA from PNA. polysaccharides, etc., was performed at 1.5” in a Tiselius cell.8 When a crude solution in the NaCl-phosphate buffer mentioned above was subjected to electrophoresis, the ascending mobility of DNA was found as -17 X lo+, that of PNA as -12.3 X KV sq. cm./v./sec., a difference in itself not adequate for the separation of sufficient amounts of DNA TABLE Purification
of Transforming
I
Preparations Types b and
from
Hemophilus
influensae,
c steps No. 1
PNA, % of DNA Protein, % of DNA Immunologically active DNA Lowest concentration ducing transformation type typr
b c
polysaccharides, of DNA
(pg./ml.)
‘% of
160-164 21-33 >30
No. 2
No. 3
<1 C2.5 <0.3
in<0.062
<0.0005
<0.0004
in the course of its rapid passage through the cell. Automatic slow compensation was, therefore, applied in order to prolong the electrophoretic separation up to 67 hr., at an electrode potential of 120 v. Owing to the high molecular weight and the high viscosity of DNA, this could be done without undue disturbance of the boundaries; even at the end of this period the DNA boundary presented itself, in fact, as a single hypersharp peak (see Fig. 1). The PNA peak flattened during this time because of diffusion, but was still sufficiently distinct for clean separation. When the DNA and PNA peaks, constantly visible owing to the compensstion, had separated by 2(r25 mm., the compensation was stopped and the DNA boundary permitted to migrate into the clean top section of the cell,’ until the 3 The instrument used was model 38 of the Perkin-Elmer Corporation, Glenbrook, Conn. Cells of a capacity of 2 or 6 ml. were employed; a special extension tube in the top section of the cell permitted the separation of larger volumes. We are grateful to Dr. D. H. Moore of this College for help and advice. 4 This step is necessary because the lower sections of the cell are contaminated by the crude viscous solution.
DNA
OF H. INFLJJENZAE
53
foot of the PNA peak nearly touched the dividing line between the cell sections. Following the separation of the sections in the usual way, the DNA fraction was collected from the top section. Up to 25% of the DNA contained in the original solution was thus obtained. The remainder was recovered from the other cell sections and resubjected to electrophoresis two to four more times. When it became too diluted, its cont,ents were precipitated with 2 vol. ethanol, dialyzed, and subjected to electrophoretic separation as before, though in a smaller cell. The combined DNA fractions from two to four electrophoresis runs (step No. 2 in Table I) were precipit,ated with 2 vol. ethanol; the resulting threads were
FIG. 1. Electrophoresis pattern (ascending) of crude transforming preparation from Hemophilus in$uenzae, type c, containing 1.82 mg. DNA/ml.; after 336 min. at 6.3 V./cm. in citrate-chloride buffer of pH 7.25, ionic strength 0.2. The faster sharp peak on the left side belongs to DNA, that on the right side to PNA. lifted and dissolved in l-2 ml. of 10% aqueous sodium chloride. For the stepwise removal of protein, the solution was shaken four times, each for 12 hr., with g vol. chloroform%-pentanol (3:l). The DNA, recovered finally in fibrous form, by the addition of 2 vol. ethanol, was dissolved in l-2 ml. of 0.16 M sodium chloride solution, to yield a clear viscous liquid. The final preparations are listed in Table I as step No. 3. All operations were carried out in the cold. RESULTS is illustrated
The progress of purification in Table I. Unless specified, ‘Ihe data refer to preparations of the transforming principles from both
54
ZAMENHOF,
LEIDY,
ALEXANDER,
FITZGERALD
AND
CHARGAFF
types b and c. It will be seen that the electrophoretic separation of DNA eliminated practically all PNA and the immunologically active polysaccharides. With respect to the latter, the limits of sensitivity of the serological reactions must, of course, be taken into account and the estimates are by necessity of no great accuracy. The final preparations were active in inducing transformations in concentrations of less than 0.0004 pg./ml. (type b) and less than 0.01 pg./ml. (type c). When the solution of the final DNA preparation of type c was dialyzed, lyophilized, and dried in 2racu.oat 60” over PaOaoperations destructive of transforming activity-the residue assayed for 96% DNA and contained N 15.75 and P 8.850/,. These results as well as those on the purine and pyrimidine composition of this preparaCon, submitted in the forthcoming paper (13), exclude the presence of major impurities in the DNA. No decrease in the transforming activity per weight unit of DNA was noted with the preceding purification and removal of proteins and other admixtures. In the case of the preparation from type b the electrophoretic separation in particular seemed, in fact, to yield a more active DNA. The results strongly suggest the importance of the DNA of Hemophdua influenzae or of a fraction contained in it for the transformation reaction under discussion. SUMMARY
The purification of DNA preparations of Hemophilus injluenzae, types b and c, endowed with transforming activity is described. The mild procedure makes use of electrophoretic separation of the DNA from contaminating PNA and polysaccharides. The final DNA preparations, free of detectable amounts of these contaminants and of proteins, were active in transformation in concentrations of less than 0.0004 pg. and 0.01 pg. DNA/ml. for types b and c, respectively. REFERENCES 1. ZAMENHOF, S., LEIDY, G., ALEXANDER. H. E., FITZGERALD, P. L., AND CEIARGAFF, E., Abstracts, p. 100. XIIth International Congress of Pure and Applied Chemistry, New York, September 1951. 2. AVERY, 0. T., MACLEOD, C. M., AND MCCARTY, M., J. Exptl. Med. 79, 137 (1944). 3. MCCARTY, M., AND AVERY, 0. T., J. Exptl. Med. 83,89 (1946). 4. ALEXANDER, H. E., AND LEIDY, G., Proc. Sot. Exptl. Biol. Med. 73,435 (1950); J. Ezptl. Med. 93, 345 (1951).
DNA
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
OF
H.
INFLUENZAE
55
SEVAG, M. G., Biochem. 2.273, 419 (1934). MCCARTY, M., AND AVERY, 0. T., J. Ezptl. Med. 83,97 (1946). KUNITZ, M., J. Gen. Physiol. 24, 15 (1940). LORING, H. S., CARPENTER, F. H., AND ROLL, P. M., J. Biol. Chem. 169, 601 (1947). SCHMIDT, G., CUBILES, R., SWARTZ, B. H., AND THANNHAUSER, S. J., J. Biol. Chem. 170, 759 (1947). MA.GASANIK, B., AND CHARGA~F, E., Biochem. et Biophys. Acta 7, 396 (1951). COHEN, S. S., J. Biol. Chem. 146, 471 (1942). CHARGAFF, E., AND ZAMENHOF, S., J. Biol. Chem. 173, 327 (1948). ZAMENHOF, S., BRAWERMAN, G., AND CHARGAFF, E., in preparation. DISCHE, Z., Mikrochemie 8, 4 (1930). MIUBAUM, W., 2. physiol. Chem. 208, 117 (1939). DIYCHE, Z., J. Biol. Chem. 181, 379 (1949).