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A minimum estimate for the length of the DNA of Escherichia coli obtained by autoradiography
J. Mol. Biol. (1962) 4, 407-409
A Minimum Estimate for the Length of the DNA of Escherichia Coli obtained by Autoradiography JOHN CAffiNS
Department of Microbiology, The Australian National University, Canberra, Australia (Received 23 January 1961) E. coli labelled with [3H]thymine was lysed in EDTA and 2 M-sucrose by dialysis against Duponol. After 20 hours the DNA of some bacteria had partly untangled and was trapped on the wall of the dialysis membrane when the dialysis chamber was drained. Autoradiography showed this DNA to be several hundred microns long, indicating a molecular weight of 109 or more. Short fragments, which would correspond to the usual molecular weight of extracted bacterial DNA, were not seen.
1. Introduction The nucleus of E. coli contains about 4 x 109 daltons of DNA (Fuerst & Stent, 1956). All this DNA replicates semiconservatively in an orderly fashion, each round of replication being completed before the next starts (Meselson & Stahl, 1958). It is dispensed semiconservatively among the descendant nuclei, only occasionally being subject to further dispersal (Forro & Wertheimer, 1960). Genetically, the bacterial nucleus is haploid (Witkin, 1951) and the genome comprises a single linkage group which can be transferred entire as a linear structure at the time of mating (Jacob & Wollman, 1958). In short, each bacterial nucleus behaves as if it contained a single molecule of DNA, by inference of some 4 x 109 molecular weight. Against this notion of a single huge DNA molecule within each nucleus is the fact that extracted bacterial DNA has always proved to be of uniformly low molecular weight. Such uniformity of molecular weight, it has been argued, could hardly be the product of random breakage at the time of extraction. Pre-existing breakage points, such as protein links, must be built into the genome at uniformly spaced points. The persuasiveness of this argument is now seen to be illusory. Large molecules of DNA, subject to shear breakage, break near the centres; the half molecules then repeat this process, and so on until some size is reached that will survive the shear being applied (Hershey & Burgi, 1960; Levinthal & Davison, 1961). It is not at all improbable that breakage of bacterial DNA should produce fairly uniform fragments. Indeed, judging from the fragility of T2 bacteriophage DNA (Davison, 1959), it is unlikely that this hypothetical molecule, which would be 20 to 40 times as large and some 2 mm long, could survive the stresses involved in many of the procedures used for determining molecular weight, let alone the violent explosion that must normally occur at the moment of bacterial lysis. An attempt has therefore been made to determine the length of bacterial DNA by autoradiography (Cairns, 1961), using an extraction procedure, known to release DNA (Meselson & Stahl, 1958) but applied in this case in such a way that all explosive forces, turbulence and shear were minimal. 407
408
JOHN CAIRNS
2. Materials and Methods Bacterium. The thymineless strain, E. coli B3 (Brenner). Medium. The glucose-ammonium medium described by Hershey (1955), supplemented with 2 p.g/ml. thymine. Preparation of labelled bacteria. E. coli B3 was grown with aeration to 1·5 x 10B/mI., centrifuged and resuspended in an equal volume of medium containing 2 p.g/ml. [3H]thymine (11·2 elm-mole). After 2 hr the viable count had risen to 3'3 x lOB/mI. This was rather less than the increase of control bacteria in cold thymine, suggesting that the presence of tritium had caused some inactivation. Lysis of bacteria. Labelled bacteria were suspended at a concentration of 5 x 105/ml. in 2 M-sucrose, 0·02 M-NaCl, 0·01 M-EDTA pH 8, with 6 p.g/ml. phenol-extracted T2 DNA. This mixture was placed in a cylindrical chamber 20 rom diameter and 2·5 rom deep, faced on one side with glass and on the other with a VM Millipore filter (50 mp. poresize). The enclosed bacteria were then dialysed at 42°C against 1% sodium lauryl sulphate (Duponol) in sucrose-NaCI-EDTA for 45 min. The Duponol was removed by dialysis, the salt concentration raised to 4 M·NaCI for I hr and then lowered to 0·04 M, EDTA being present throughout. After a further 16 hr, the chamber was stood on its side, punctured in two places, and drained slowly on to filter paper. The Millipore filter was allowed to dry in situ. The reasoning behind this sequence was as follows. Duponol and EDTA liberate DNA from E. coli in such a form that much of any attendant protein (and this includes the hypothetical linkers) can be separated by high salt concentration when the mixture is banded in CsCI (Meselson & Stahl, 1958). Here no removal of any protein freed by high salt concentration can be expected, but the considerable excess of carrier DNA and the very low concentration of bacterial DNA (0,01 p.g/mI.) should tend to prevent bacterial DNA and protein from re-uniting when the salt concentration, after being raised, is lowered again. To avoid explosion of the bacteria at the moment of lysis, lysis was carried out in 2 M-sucrose. Autoradiography. The Millipore filters were stuck to microscope slides with a cement, overlaid with Kodak autoradiographic stripping film; AR 10, and stored at 4°C over CaS0 4 in an atmosphere of CO 2 , After exposure the film was developed with Kodak DI9b for 20 min at 16°C.
3. Results The results can be seen in Plate I and may be summarized as follows. (1) Although lysis has occurred (compare the inset autoradiograph of unlysed bacteria), the DNA of most bacteria has not unravelled enough to display any features of its form. This is not surprising since the only disentangling forces were Brownian movement, convection currents and finally the slow passage of the meniscus across the filter when the dialysis chamber was drained. (2) The DNA extracted from bacteria by normal methods has a sedimentation coefficient of around 25 s (Marmur, 1961), indicating a molecular weight of 20 to 30 X 106 (Rubinstein, Thomas & Hershey, 1961) and a length of some 10 p.. No such pieces are to be seen in the autoradiograph. If bacterial DNA is truly divided into such lengths by linkers, then the extraction procedure used here has totally failed to dissolve these hypothetical links even though virtually the same procedure invariably breaks them when applied without precautions against shear; this seems highly improbable. If, on the other hand, such lengths are a casual product of turbulence and shear per se, it is reasonable that there should have been little fragmentation. (3) A few pieces of DNA can be seen which have two identifiable ends and can therefore be measured. Their lengths range from 25 p. upwards.
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PLATE I. E. coli D N A, labelled with [3H ]th y m ine (11·2 elm-m ol e), extract ed with D u ponol and EDTA and collected on a Milli pore filt er at a concentration of 0· 01 }Lg/m J. The inset p ho t ograp h shows. at t he sa me magn ifica tio n, an au t oradiogr aph of u nlys ed bacter ia. ThA aut oradi ographic ex p osure was 70 days. Th e scale shows 100 JL.
AUTORADIOGRAPHY OF E. GOLI DNA
409
(4) More commonly the DNA has partly untangled to display a long unbroken thread; two such threads, both about 400 /-L long, are shown in the Plate. Such threads may cross others, but they show no branches. As the concentration of untangled labelled DNA must be much less than 0·01 /-Lg!ml., they can hardly represent artificial end-to-end aggregation of smaller pieces. Until the existence in DNA of non-nucleic acid links has been demonstrated, it is probably legitimate to think of these threads as molecules. From their length, they must have a molecular weight of around 109 • As it has not been possible to identify with certainty both ends of any of these long molecules, this must be a minimum estimate of the molecular weight of bacterial DNA. Note added in proof. Since this paper was written, Kleinschmidt, Lang & Kahn (1961) have produced beautiful electron micrographs showing that protoplasts of M. lysodeikticus, lysed at an air-water interface, release their deoxyribonucleoprotein in the form of a tangled skein which has no visible free ends. Thus two dissimilar procedures suggest that bacterial DNA may exist as a single molecule. REFERENCES Cairns, J. (1961). J. Mol. Biol. 3, 756. Davison, P. F. (1959). Proc. Nat. Acad. Sci., Wash. 45, 1560. Forro, F. & Wertheimer, S. A. (1960), Biochim. biophys. Acta, 40, 9. Fuerst, C. R. & Stent, G. S. (1956). J. Gen. Physiol. 40, 73. Hershey, A. D. (1955). Virology, 1, 108. Hershey, A. D. & Burgi, E. (1960). J. Mol. Biol. 2, 143. Jacob, F. & Wollman, E. L. (1958). Symp. Soc. Exp. Biol. 12, 75. Kleinschmidt, A., Lang, D. & Kahn, R. K. (1961). Z. Naturf. 16b, 730. Levinthal, C. & Davison, P. F. (1961). J. Mol. Biol. 3, 674. Marmur, J. (1961). J. Mol. Biol. 3, 208. Meselson, M. & Stahl, F. W. (1958). Proc. Nat. Acad. Sci., Wash. 44, 671. Rubinstein, 1., Thomas, C. A. & Hershey, A. D. (1961). Proc, Nat. Acad. Sci., }Vash. 47, 1113. Witkin, E. M. (1951). ColdSpr. Barb. Symp. Quant. Biol. 16,357.
A Minimum Estimate for the Length of the DNA of Escherichia Coli obtained by Autoradiography JOHN CAffiNS
Department of Microbiology, The Australian National University, Canberra, Australia (Received 23 January 1961) E. coli labelled with [3H]thymine was lysed in EDTA and 2 M-sucrose by dialysis against Duponol. After 20 hours the DNA of some bacteria had partly untangled and was trapped on the wall of the dialysis membrane when the dialysis chamber was drained. Autoradiography showed this DNA to be several hundred microns long, indicating a molecular weight of 109 or more. Short fragments, which would correspond to the usual molecular weight of extracted bacterial DNA, were not seen.
1. Introduction The nucleus of E. coli contains about 4 x 109 daltons of DNA (Fuerst & Stent, 1956). All this DNA replicates semiconservatively in an orderly fashion, each round of replication being completed before the next starts (Meselson & Stahl, 1958). It is dispensed semiconservatively among the descendant nuclei, only occasionally being subject to further dispersal (Forro & Wertheimer, 1960). Genetically, the bacterial nucleus is haploid (Witkin, 1951) and the genome comprises a single linkage group which can be transferred entire as a linear structure at the time of mating (Jacob & Wollman, 1958). In short, each bacterial nucleus behaves as if it contained a single molecule of DNA, by inference of some 4 x 109 molecular weight. Against this notion of a single huge DNA molecule within each nucleus is the fact that extracted bacterial DNA has always proved to be of uniformly low molecular weight. Such uniformity of molecular weight, it has been argued, could hardly be the product of random breakage at the time of extraction. Pre-existing breakage points, such as protein links, must be built into the genome at uniformly spaced points. The persuasiveness of this argument is now seen to be illusory. Large molecules of DNA, subject to shear breakage, break near the centres; the half molecules then repeat this process, and so on until some size is reached that will survive the shear being applied (Hershey & Burgi, 1960; Levinthal & Davison, 1961). It is not at all improbable that breakage of bacterial DNA should produce fairly uniform fragments. Indeed, judging from the fragility of T2 bacteriophage DNA (Davison, 1959), it is unlikely that this hypothetical molecule, which would be 20 to 40 times as large and some 2 mm long, could survive the stresses involved in many of the procedures used for determining molecular weight, let alone the violent explosion that must normally occur at the moment of bacterial lysis. An attempt has therefore been made to determine the length of bacterial DNA by autoradiography (Cairns, 1961), using an extraction procedure, known to release DNA (Meselson & Stahl, 1958) but applied in this case in such a way that all explosive forces, turbulence and shear were minimal. 407
408
JOHN CAIRNS
2. Materials and Methods Bacterium. The thymineless strain, E. coli B3 (Brenner). Medium. The glucose-ammonium medium described by Hershey (1955), supplemented with 2 p.g/ml. thymine. Preparation of labelled bacteria. E. coli B3 was grown with aeration to 1·5 x 10B/mI., centrifuged and resuspended in an equal volume of medium containing 2 p.g/ml. [3H]thymine (11·2 elm-mole). After 2 hr the viable count had risen to 3'3 x lOB/mI. This was rather less than the increase of control bacteria in cold thymine, suggesting that the presence of tritium had caused some inactivation. Lysis of bacteria. Labelled bacteria were suspended at a concentration of 5 x 105/ml. in 2 M-sucrose, 0·02 M-NaCl, 0·01 M-EDTA pH 8, with 6 p.g/ml. phenol-extracted T2 DNA. This mixture was placed in a cylindrical chamber 20 rom diameter and 2·5 rom deep, faced on one side with glass and on the other with a VM Millipore filter (50 mp. poresize). The enclosed bacteria were then dialysed at 42°C against 1% sodium lauryl sulphate (Duponol) in sucrose-NaCI-EDTA for 45 min. The Duponol was removed by dialysis, the salt concentration raised to 4 M·NaCI for I hr and then lowered to 0·04 M, EDTA being present throughout. After a further 16 hr, the chamber was stood on its side, punctured in two places, and drained slowly on to filter paper. The Millipore filter was allowed to dry in situ. The reasoning behind this sequence was as follows. Duponol and EDTA liberate DNA from E. coli in such a form that much of any attendant protein (and this includes the hypothetical linkers) can be separated by high salt concentration when the mixture is banded in CsCI (Meselson & Stahl, 1958). Here no removal of any protein freed by high salt concentration can be expected, but the considerable excess of carrier DNA and the very low concentration of bacterial DNA (0,01 p.g/mI.) should tend to prevent bacterial DNA and protein from re-uniting when the salt concentration, after being raised, is lowered again. To avoid explosion of the bacteria at the moment of lysis, lysis was carried out in 2 M-sucrose. Autoradiography. The Millipore filters were stuck to microscope slides with a cement, overlaid with Kodak autoradiographic stripping film; AR 10, and stored at 4°C over CaS0 4 in an atmosphere of CO 2 , After exposure the film was developed with Kodak DI9b for 20 min at 16°C.
3. Results The results can be seen in Plate I and may be summarized as follows. (1) Although lysis has occurred (compare the inset autoradiograph of unlysed bacteria), the DNA of most bacteria has not unravelled enough to display any features of its form. This is not surprising since the only disentangling forces were Brownian movement, convection currents and finally the slow passage of the meniscus across the filter when the dialysis chamber was drained. (2) The DNA extracted from bacteria by normal methods has a sedimentation coefficient of around 25 s (Marmur, 1961), indicating a molecular weight of 20 to 30 X 106 (Rubinstein, Thomas & Hershey, 1961) and a length of some 10 p.. No such pieces are to be seen in the autoradiograph. If bacterial DNA is truly divided into such lengths by linkers, then the extraction procedure used here has totally failed to dissolve these hypothetical links even though virtually the same procedure invariably breaks them when applied without precautions against shear; this seems highly improbable. If, on the other hand, such lengths are a casual product of turbulence and shear per se, it is reasonable that there should have been little fragmentation. (3) A few pieces of DNA can be seen which have two identifiable ends and can therefore be measured. Their lengths range from 25 p. upwards.
"
,
.
..
, "
,
•
•
PLATE I. E. coli D N A, labelled with [3H ]th y m ine (11·2 elm-m ol e), extract ed with D u ponol and EDTA and collected on a Milli pore filt er at a concentration of 0· 01 }Lg/m J. The inset p ho t ograp h shows. at t he sa me magn ifica tio n, an au t oradiogr aph of u nlys ed bacter ia. ThA aut oradi ographic ex p osure was 70 days. Th e scale shows 100 JL.
AUTORADIOGRAPHY OF E. GOLI DNA
409
(4) More commonly the DNA has partly untangled to display a long unbroken thread; two such threads, both about 400 /-L long, are shown in the Plate. Such threads may cross others, but they show no branches. As the concentration of untangled labelled DNA must be much less than 0·01 /-Lg!ml., they can hardly represent artificial end-to-end aggregation of smaller pieces. Until the existence in DNA of non-nucleic acid links has been demonstrated, it is probably legitimate to think of these threads as molecules. From their length, they must have a molecular weight of around 109 • As it has not been possible to identify with certainty both ends of any of these long molecules, this must be a minimum estimate of the molecular weight of bacterial DNA. Note added in proof. Since this paper was written, Kleinschmidt, Lang & Kahn (1961) have produced beautiful electron micrographs showing that protoplasts of M. lysodeikticus, lysed at an air-water interface, release their deoxyribonucleoprotein in the form of a tangled skein which has no visible free ends. Thus two dissimilar procedures suggest that bacterial DNA may exist as a single molecule. REFERENCES Cairns, J. (1961). J. Mol. Biol. 3, 756. Davison, P. F. (1959). Proc. Nat. Acad. Sci., Wash. 45, 1560. Forro, F. & Wertheimer, S. A. (1960), Biochim. biophys. Acta, 40, 9. Fuerst, C. R. & Stent, G. S. (1956). J. Gen. Physiol. 40, 73. Hershey, A. D. (1955). Virology, 1, 108. Hershey, A. D. & Burgi, E. (1960). J. Mol. Biol. 2, 143. Jacob, F. & Wollman, E. L. (1958). Symp. Soc. Exp. Biol. 12, 75. Kleinschmidt, A., Lang, D. & Kahn, R. K. (1961). Z. Naturf. 16b, 730. Levinthal, C. & Davison, P. F. (1961). J. Mol. Biol. 3, 674. Marmur, J. (1961). J. Mol. Biol. 3, 208. Meselson, M. & Stahl, F. W. (1958). Proc. Nat. Acad. Sci., Wash. 44, 671. Rubinstein, 1., Thomas, C. A. & Hershey, A. D. (1961). Proc, Nat. Acad. Sci., }Vash. 47, 1113. Witkin, E. M. (1951). ColdSpr. Barb. Symp. Quant. Biol. 16,357.