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Reconstitution of nucleohistone from homologous and heterologous components
J. Mol. Bid.
(1968) 38, 249-250
Reconstitution of Nucleohistone from Homologous and Heterologous Components The mode of interaction of histones with DNA in the chromosomes of higher organisms is still an unsolved problem. Studies on the binding of histones (Chargaff, Crampton & Lipschitz, 1953; Brown & Watson, 1953; Ohba, 1966), and synthetic polyamino acids (Spit& Lipschitz & Chargaff, 1955; Leng & Felsenfeld, 1966) indicate that in the right circumstances, the polybase can recognize and attach itself preferentially to AST-rich regions of the DNA. Furthermore, it is established that histones, but not to date synthetic polypeptides or protamines, cause the DNA to assume a characteristic native conformation (Pardon, 1966; Pardon, Wilkins & Richards, 1967), which produces a characteristic X-ray diffraction pattern, consisting of a series of low-angle reflections at 55, 37, 27 and 22 A (Wilkins, Zubay & Wilson, 1959; Pardon & Wilkins, results to be published). The ability of histone and DNA from calf thymus to recombine and reproduce this native nucleohistone conformation and X-ray diffraction pattern (Zubay & Wilkins, 1964) leads one to the fundamental question of whether this conformational specificity resides only in some general feature of the histone molecules as a group, or whether it requires that the individual histone molecules return to their precise locations on the DNA, defined by a specific base-sequence. An attempt to clarify this problem was made by preparing heterologous nucleohistone in which histones, separated from one source, were added to DNA from another. The characteristic X-ray diffraction pattern was used as a criterion for the presence of the native nucleohistone conformation. Whole histone was extracted from fresh, frozen, calf thymus by method 2 of Zubay & Wilkins (1962). In some experiments the pH was reduced from 1.6 to O-7 to ensure complete removal of histones (Murray, 1966), with similar results. The solution was not concentrated, although partial lyophilisation did not affect the results. The DNA content was shown to be less than 0.25% with the diphenylamine reagent (Burton, 1956). DNA from calf thymus (Sigma Chemical Corp.) and salmon sperm (Sigma Chemical Corp. and gift) and T7 virus (gift) were dissolved in 0.01 M-phosphate buffer to a concentration of approximately 1 mg/ml. at pH 5. Each solution was adjusted to 2.6 M-Nacl, 0.1 M-phosphate buffer at pH 5 by adding salt. A histone solution was added with stirring to each DNA solution (histone-DNA was approximately 1.1: 1 w/w), and dialysed overnight against 0.15 M-NaC1 at pH 7. The precipitate was partially oriented by stroking. Fibre X-ray diffraction photographs were obtained using pin-hole collimating cameras (Langridge, Wilson, Hooper, Wilkins & Hamilton, 1960) and an Elliott toroidal camera (Elliott, 1965). X-ray diffraction of the homologous nucleoli&tone, as before (Zubay & Wilkins, 1964), and the heterologous salmon and T7 viral DNA complexes, produced reflections at approximately 55, 37,27 and 22 A, characteristic of native nucleohistones at 98% relative humidity (Wilkins et al., 1959; and unpublished observations). Plate I shows an X-ray diffraction pattern of heterologous nucleohistone (salmon DNA and calf thymus histone) at 98% relative humidity. 249
250
R. A. GARRETT
As judged by c~-c (a more sensitive met.hod of detection was not advanbageous because the rclativc intrnsitics vary slightly with the slit content of the fibre spccimens), the relative intensit,ies of the raflcctions were the same, nt OS% rktivr humid&y, as those produced by both reconstituted homoIogons nucleohistonc samples, and a large number of native calf thymus nucleohistonc samples (rrnpublished observations), prepared from both soluble and gel nuclcohistnnc which was extmcted using a modified Zubay & Doty extraction procedure(1’3%). Commercial DNA (Sigma Chemiesl Corp.) nucleohist.one complexes produced cont.inuous scatter at a.ngles smaller than about 30 A, which may have been causrd by the presence of a small proportion of uncoiled DhX molecules. Salmon Dl\iil gave a mixture of A and R patterns at. 9s0& relative humidity and produced nonc of these rrflcetions. Calf thymus histones alone produced a \veak diffuse ring at 35 a (Zubay & Wilkins, 1962). These results indicate t,hat almost all of the Dh’B has returned to its native conformat’ion and 0111:concludes that the characteristic conformation of the DX% in nucleohistonc is determined by t,he native hi&one structure and not by any specific base-sequence association bctwecn cndogcnous hi&one and DNS. I am indebted to Professor 21. H. F. Wilkins for discussion and help with obtaining diffraction patterns, to Dr W. Gratzer for ndvice, to Professor Sir John Randall for support, to Professor M. Bow for a gift of T7 viral DNA and to Dr S. Amott for a gift of salmon sperm DNA (pcapared by Dr L. D. Hamilton). I am a rccipicnt of a Science Resea.reh Council research studentship.
Department of Biophysics King’s Coltego, 26-29 Drury Lane London, W.C.2, England
ROGER A. G-4umwrt
Received 21 June 1968, and in rovised form 4 September 1968 REFERENCES Brown, 0. L. & Watson, M. (1953). Nature, 172, 339. Burton, K. (1956). B&hem. J. 62, 315. Chargaff, E., Crampton, C. F. & Lipschitz, R. (1953). *Valure, 172, 289. Elliott, A. (1965). J. Sci. I&r. 42, 312. Langridge, R., Wilson, H. R., Hoopor, C. W., Wilkins, M. H. F. & Hamilton, (1960).
J. WOE. Bid.
L. D.
2, 19.
Lcng, M. & Folsenfeld, C. (1966). I%c. Aiat. Ad. Sci., Wash. Murray, K. (1966). J. MOE. Biol. 15, 409. Ohba, Y. (1966). Biuchim. biophya. A&, 124, 84. Pardon, J. F. (1966). Ph.D. thesis, University of London.
56, 13!2L
Pardon, Spit’nik, Wilkins, Zubay,
J. F., Wilkins, M. H. F. & Richards, 13. M. (1967). Nature, 215, 508. P., Lipschitz, R. & Chargaff, E. (1955). J. Bid. Chem. 215, 765. &I. H. F., Zubay, G. &Wilson, H, R. (1959). J. afoE. B&Z. 1, 179. 0. 85 Doty, P. (1959). J. Mol. Bid. 1, 1. Zubay, G. & Wilkins, M. H. F. (1962). J. MOE. Bid. 4, 444. Zubay, G. & Wilkins, M. H. F. (1964). J. Mol. Bid. 9, 246.
7 Present Netherlands.
address:
Department
of Physiological
Chemistry,
State Uniniveraity
of &den,
The
(1968) 38, 249-250
Reconstitution of Nucleohistone from Homologous and Heterologous Components The mode of interaction of histones with DNA in the chromosomes of higher organisms is still an unsolved problem. Studies on the binding of histones (Chargaff, Crampton & Lipschitz, 1953; Brown & Watson, 1953; Ohba, 1966), and synthetic polyamino acids (Spit& Lipschitz & Chargaff, 1955; Leng & Felsenfeld, 1966) indicate that in the right circumstances, the polybase can recognize and attach itself preferentially to AST-rich regions of the DNA. Furthermore, it is established that histones, but not to date synthetic polypeptides or protamines, cause the DNA to assume a characteristic native conformation (Pardon, 1966; Pardon, Wilkins & Richards, 1967), which produces a characteristic X-ray diffraction pattern, consisting of a series of low-angle reflections at 55, 37, 27 and 22 A (Wilkins, Zubay & Wilson, 1959; Pardon & Wilkins, results to be published). The ability of histone and DNA from calf thymus to recombine and reproduce this native nucleohistone conformation and X-ray diffraction pattern (Zubay & Wilkins, 1964) leads one to the fundamental question of whether this conformational specificity resides only in some general feature of the histone molecules as a group, or whether it requires that the individual histone molecules return to their precise locations on the DNA, defined by a specific base-sequence. An attempt to clarify this problem was made by preparing heterologous nucleohistone in which histones, separated from one source, were added to DNA from another. The characteristic X-ray diffraction pattern was used as a criterion for the presence of the native nucleohistone conformation. Whole histone was extracted from fresh, frozen, calf thymus by method 2 of Zubay & Wilkins (1962). In some experiments the pH was reduced from 1.6 to O-7 to ensure complete removal of histones (Murray, 1966), with similar results. The solution was not concentrated, although partial lyophilisation did not affect the results. The DNA content was shown to be less than 0.25% with the diphenylamine reagent (Burton, 1956). DNA from calf thymus (Sigma Chemical Corp.) and salmon sperm (Sigma Chemical Corp. and gift) and T7 virus (gift) were dissolved in 0.01 M-phosphate buffer to a concentration of approximately 1 mg/ml. at pH 5. Each solution was adjusted to 2.6 M-Nacl, 0.1 M-phosphate buffer at pH 5 by adding salt. A histone solution was added with stirring to each DNA solution (histone-DNA was approximately 1.1: 1 w/w), and dialysed overnight against 0.15 M-NaC1 at pH 7. The precipitate was partially oriented by stroking. Fibre X-ray diffraction photographs were obtained using pin-hole collimating cameras (Langridge, Wilson, Hooper, Wilkins & Hamilton, 1960) and an Elliott toroidal camera (Elliott, 1965). X-ray diffraction of the homologous nucleoli&tone, as before (Zubay & Wilkins, 1964), and the heterologous salmon and T7 viral DNA complexes, produced reflections at approximately 55, 37,27 and 22 A, characteristic of native nucleohistones at 98% relative humidity (Wilkins et al., 1959; and unpublished observations). Plate I shows an X-ray diffraction pattern of heterologous nucleohistone (salmon DNA and calf thymus histone) at 98% relative humidity. 249
250
R. A. GARRETT
As judged by c~-c (a more sensitive met.hod of detection was not advanbageous because the rclativc intrnsitics vary slightly with the slit content of the fibre spccimens), the relative intensit,ies of the raflcctions were the same, nt OS% rktivr humid&y, as those produced by both reconstituted homoIogons nucleohistonc samples, and a large number of native calf thymus nucleohistonc samples (rrnpublished observations), prepared from both soluble and gel nuclcohistnnc which was extmcted using a modified Zubay & Doty extraction procedure(1’3%). Commercial DNA (Sigma Chemiesl Corp.) nucleohist.one complexes produced cont.inuous scatter at a.ngles smaller than about 30 A, which may have been causrd by the presence of a small proportion of uncoiled DhX molecules. Salmon Dl\iil gave a mixture of A and R patterns at. 9s0& relative humidity and produced nonc of these rrflcetions. Calf thymus histones alone produced a \veak diffuse ring at 35 a (Zubay & Wilkins, 1962). These results indicate t,hat almost all of the Dh’B has returned to its native conformat’ion and 0111:concludes that the characteristic conformation of the DX% in nucleohistonc is determined by t,he native hi&one structure and not by any specific base-sequence association bctwecn cndogcnous hi&one and DNS. I am indebted to Professor 21. H. F. Wilkins for discussion and help with obtaining diffraction patterns, to Dr W. Gratzer for ndvice, to Professor Sir John Randall for support, to Professor M. Bow for a gift of T7 viral DNA and to Dr S. Amott for a gift of salmon sperm DNA (pcapared by Dr L. D. Hamilton). I am a rccipicnt of a Science Resea.reh Council research studentship.
Department of Biophysics King’s Coltego, 26-29 Drury Lane London, W.C.2, England
ROGER A. G-4umwrt
Received 21 June 1968, and in rovised form 4 September 1968 REFERENCES Brown, 0. L. & Watson, M. (1953). Nature, 172, 339. Burton, K. (1956). B&hem. J. 62, 315. Chargaff, E., Crampton, C. F. & Lipschitz, R. (1953). *Valure, 172, 289. Elliott, A. (1965). J. Sci. I&r. 42, 312. Langridge, R., Wilson, H. R., Hoopor, C. W., Wilkins, M. H. F. & Hamilton, (1960).
J. WOE. Bid.
L. D.
2, 19.
Lcng, M. & Folsenfeld, C. (1966). I%c. Aiat. Ad. Sci., Wash. Murray, K. (1966). J. MOE. Biol. 15, 409. Ohba, Y. (1966). Biuchim. biophya. A&, 124, 84. Pardon, J. F. (1966). Ph.D. thesis, University of London.
56, 13!2L
Pardon, Spit’nik, Wilkins, Zubay,
J. F., Wilkins, M. H. F. & Richards, 13. M. (1967). Nature, 215, 508. P., Lipschitz, R. & Chargaff, E. (1955). J. Bid. Chem. 215, 765. &I. H. F., Zubay, G. &Wilson, H, R. (1959). J. afoE. B&Z. 1, 179. 0. 85 Doty, P. (1959). J. Mol. Bid. 1, 1. Zubay, G. & Wilkins, M. H. F. (1962). J. MOE. Bid. 4, 444. Zubay, G. & Wilkins, M. H. F. (1964). J. Mol. Bid. 9, 246.
7 Present Netherlands.
address:
Department
of Physiological
Chemistry,
State Uniniveraity
of &den,
The