- Email: [email protected]
Differential accessibility of DNA in extended and condensed chromatin to pancreatic DNase I
5 18 Preliminary notes
erythrocyte heterokaryons [8, 181.Although 16. Ege, T, Carlsson, S-A & Ringertz, N R, Exptl cell res 69 (1971) 472. the accumulation of T antigen in chick 17. Dupuy-Coin, A-M, Ege, T, Bouteille, M & Ringertz, N R. In preparation. erythrocyte nuclei closely paralleled nuclear 18. Appels, R, Tallroth, E, Appels, D M & Ringertz, enlargement this is partly coincidental beN R, Exptl cell res 92 (1975) 70. causeprevious results obtained by Koprowski 19. Koprowski, H, Jensen, F C & Steplewski, Z, Proc natl acad sci US 58 (1967) 127. et al. [19,20] show that the T antigen can also 20. Steplewski. Z. Knowles. B & Konrowski. H. ’ ’ Proc natl acad sci US 59’(1968) 769.‘ migrate into non-transformed nuclei which are already active before fusion. The migra- Received February 17, 1975 tion of T antigen into reactivating chick erythrocyte nuclei is of interest not only as a part of the reactivation process but also because chick cells cannot be transformed or Differential accessibility of DNA in extended infected with SV40. Nuclear uptake of SV40 and condensedclwomatin to pancreatic DNase I specified T antigen therefore is independent and M. G. WEAVER, of the permissivenessand transformability of G. D. BURKHOLDER Department of Anatomy, College of Medicine, Unithe mammalian cell. versity of Saskatchewan, Saskatoon, Saskatchewan S7N 0 WO, Canada This investigation was supported by grants from the Swedish Cancer Society and Sigrid Juselius Foundation. The authors wish to thank Dr J. van der Noordaa, University of Amsterdam, Holland, for the generous gift of T antisera.
References 1. Cameron, I & Prescott, D, Exptl cell res 56 (1969) 406. 2. Ringertz, N R & Bolund, L. The cell nucleus (ed H Busch) vol. III, p. .417. Academic Press, New York (1974). Harris, H, J cell sci 2 (1967) 23. Nucleus and cytoplasm. Oxford Univ Press, oxford, UK (1968). Bolund, L, Ringer& N R & Harris, H, J cell sci 4 (1969) 71. Ringer& N R & Bolund, L, Int rev exp path01 (ed G W Richter & M A Epstein) vol. 13, p. 83. Academic Press, New York (1974). 7. Ringertz N R, Carlsson, S-A Ege, T & Bolund, L, Proc natl acad sci US 68 (1971) 3228. 8. Appels, R, Bolund, L & Ringertz N R, J mol biol 87 (1974) 339. 9. Appels, R & Ringertz N R, Current topics of dev biol (ed A MONOY & M Moscona). Academic Press, New York (1975). In press. Naha, P M. Exntl cell res 80 (1973) 467. :?I Butel, J S, Terethia S S & Melnick, J L, Adv in cant res (ed G Klein) vol. 15, p. 55. Academic Press, New York (1972). 12. Girardi A J, Jensen, F C & Koprowski, H, J cell camp physiol 65 (1965) 69. 13. Carlsson, S-A, Savage, R E & Ringertz N R, Nature 228 (1970) 869. 14. Harris. H. Watkins. J F. Ford. C E & Schoefl. G I, J’cellsci 1(1966) 1.. 15. Stenman, S, Zeuthen, J & Ringertz, N R, Int j cancer res (1975). 15 (1975) 547. Exptl CeN Res 92 (1975)
Summary. Pancreatic DNase I has been used to study the interaction between DNA and chromosomal proteins in extended and condensed chromatin fractions isolated from mouse and Chinese hamster livers. It was found that DNase digests extended chromatin at a faster rate than condensed chromatin, and the evidence suggests that the chromosod proteins are more tightly complexed to the DNA in condensed than in -extended -chromatin. This difference in DNA-protein interaction in extended and condensed cbromatin may be related to the functional difference which characterizes these fractions, and might be one of the factors underlying the production of bands on metaphase chromosomes.
Deoxyribonuclease (DNase) has served as a useful probe for investigating the structural properties of chromatin [l, 7, 12, 13, 17-19, 221. Purified DNA is rapidly degraded by DNase, however the DNA in chromatin is afforded a limited measure of protection against nuclease attack, primarily as a result of the association of chromosomal proteins with the DNA [17, 221.The rate of digestion of chromatin DNA in the presence of pancreatic DNase I is dependent on the degree of binding of proteins to the DNA [22]. Chromatin in which the DNA is loosely associated with protein will be digested at a faster rate than chromatin in which the DNA is more tightly associatedwith protein.
Preliminary notes
It is therefore possible to study relative rates of digestion as a means to distinguish relative differences in chromatin structure. Several methods currently exist for separating chromatin into presumptive euchromatin (extended) and heterochromatin (condensed) fractions [II, 16, 20, 231. We have used one of these methods to obtain extended and condensed fractions from mouse and Chinese hamster livers, and have assayed the degree of complexing between DNA and chromosomal proteins in these fractions using pancreatic DNase I. Materials and Methods Liver nuclei were isolated from Chinese hamsters (Cricetulus griseus) or CsH mice by the method of Chauveau et al. [6], as modified by Comings [lo]. The livers of several fasted animals were perfused with ice-cold 0.01 M Tris buffer, pH 8.0, containing 3 x 1O-JM MgC12, 1O-8 M NaHS03, and 1O-4M CdSOd. Subsequent procedures were performed at 4°C. After mincing, the livers were homogenized in 2.4 M sucrose, 3.3 x 1O-8 M CaCIZ, and 1O-8 M NaHSO, (SCN), using a teflon homogenizer. When most of the cells were broken, the homogenate was filtered through cheesecloth and layered on top of a 4th tube volume of SCN. The interface between the two layers was mixed to form a crude gradient, and the tubes were centrifuged at 40080 g for 1 h. The nuclear pellet was subsequently washed according to the following schedule: 1 x in 0.01 M Tris buffer, pH 7.0, 5 x 1O-s M MgCl,, 3 x 1O-s M CaCl,, lo-* M NaHS03, 1O-4 M CdSOI, 0.14 M NaCl, 1 pg/ml soybean trypsin inhibitor (SW buffer) + lo-* M phenylmethylsulfonylfluoride (Calbiochem); ’ 3 x in SW buffer alone; 1 x in SW buffer + 0.05 % Triton X-100; 1 x in SW buffer alone. The nuclei were pelleted at 400 g for 5 min after each wash. The isolation of chromatin subfractions was a modification of the method of Comings [lo, 161. Washed nuclei were resuspended in 0.34 M sucrose, 0.1 pg/ml soybean trypsin inhibitor, 0.01 M Tris buffer, pH 7.0, and were left on ice for 15 min. The nuclear suspension was then sonicated with a Branson W-140 sonicator (standard horn; setting no. 8), using 10 set bursts, until phase contrast microscopy indicated that the majority of the nuclei were ruptured. Centrifugation at 400 g for 5 min pelleted unbroken nuclei, and chromatin fractions were obtained from the supernatant by differential centrifugation. The pellet obtained by centrifugation at 4000 g for 20 min contained the condensed chromatin fraction. The supematant was then centrifuged at 20 000 g for 20 min to yield the intermediate chromatin fraction, and the supernatant was recentrifuged at 100000 g for 30 min. The resulting pellet was taken as the extended chromatin fraction I, and the supematant was
5 19
the extended chromatin fraction 11. All fractions were dialyzed overnight against 0.01 M Tris buffer, nH 7.0. The purity of the chromatin fractions was determined on a morphological basis, using electron microscopy. The condensed fraction also contained nucleoli, and appeared slightly contaminated by extended chromatin. The extended chromatin fractions were extremely pure, containing little or no condensed chromatin. The intermediate fraction was a mixture of condensed and extended chromatin. DNA and protein estimations were determined using the Burton [5] and Lowry [14] assays, respectively. The protein/DNA ratios of the mouse chromatin fractions (mean of 5 determinations) were as follows: condensed, 2.36; intermediate, 2.26; extended I, 1.91; extended II, 1.65. Each dialyzed chromatin fraction was adjusted with 0.01 M Tris buffer, pH 7.0, to contain 25 pig/ml equivalent of DNA, as determined by the absorbance at 260 nm. The chromatin fractions were then warmed to 37°C and MgC& and bovine pancreatic DNase 1 (Worthington) were added in sequence to final concentrations of 3 x lO-s M and 5 pg/ml, respectively. The fractions were incubated in a shaker bath (37”C), and 1 ml samples were removed at various times thereafter. The reaction was stopped and undigested chromatin in each sample was precipitated by the addition of ice-cold perchloric acid to a final concentration of 0.25 M. The samples were left on ice for 30 min and then centrifuged at 37 000 g for 15 min to pellet undigested chromatin. The amount of digested DNA in each supernatant was quantitated by measurement of the absorbance at 260 nm. As a control, DNA was isolated from each of the chromatin fractions using the method of Marmur [15]. This purified DNA was exposed to DNase I using the same procedure as for the chromatin samples.
Results
The percentage of DNA digested in the chromatin and purified DNA fractions exposed to DNase was plotted as a function of time to give the rate of digestion (figs 1 and 2). All of the chromatin fractions exhibited two steps in their digestion rate: a fast initial rate, with 40-65 % of the DNA being digested during the first 15 min, and a slower rate of digestion thereafter. Of significance was the finding that the rates of digestion differed in the various chromatin fractions. Extended chromatin was digested at a faster rate than condensed chromatin in both the mouse and Chinese hamster (figs 1 and 2), so that, at all sampling times, a higher Exptl Cell Res 92 (1975)
520 Preliminary notes
These DNA fractions were digested at a uniform rate, and there were no differences in digestion rates comparable to their chromatin counterparts. ‘Discussion
In both the mouse and Chinese hamster, DNA in extended chromatin was more sensitive to the action of DNase than that in condensed chromatin, and the intermediate chromatin fraction generally appeared to have an intermediate sensitivity (figs I and 2). This differential sensitivity of the chromatin fractions to DNase digestion was not inherent in the DNA each contained, since Fig. I. Abscissa: time (min); ordinate: % DNA DNA samples purified from the chromatin Condensed chromatin fraction; digested. O---O, fractions did not differ in their rate of n-.-A, intermediate chromatin fraction, 0. * 0, extended chromatin fraction I. DNA isolated from digestion. The difference in the rates of condensed chromatin (O-O); intermediate chrodigestion of the chromatin fractions must matin fraction 1 (H-W). Rate of digestion of chromatin fractions isolated therefore be related to the association of the from mouse liver. Chromatin fractions or Durified chromosomal proteins with the DNA, such DNA samples were adjusted to contain 25 pg/ml e$uivalent of DNA, and the assays were performed that the DNA in condensed chromatin is with pancreatic DNase I at 5 pg/ml. See text for protected from DNase attack to a greater details. extent than that in extended chromatin. There appear to be two possible ways in percentage of DNA was digested in extended which this protection might be achieved: compared to condensed chromatin. Eight (1) The DNA in condensed chromatin may separate experiments have been conducted be more resistant to the action of DNase with isolated mouse chromatin fractions, simply becausethis chromatin is more folded and three with Chinese hamster fractions, all than the extended chromatin. It is easy to with the same result. envisage that DNA would be less accessible, The intermediate chromatin fraction was to DNase, within a mass of tightly folded somewhat variable in its rate of digestion, chromatin than in relatively unfolded chroranging from a rate similar to that of con- matin. (2) The chromosomal proteins may densed chromatin (fig. 1) to an intermediate be more tightly complexed to the DNA in condensed compared to extended chromatin. rate of digestion (fig. 2). This variability presumably reflects the degree of inter- As a result, the DNA in condensedchromatin mixing of condensed and extended chromatin would be more inaccessible to DNase than that in extended chromatin, and would in this fraction. Purified DNA, isolated from the extended, therefore be afforded a greater measure of intermediate, and condensed chromatin frac- resistance to DNase attack. Additional work tions, and exposed to DNase using the same is in progress to distinguish between these conditions as for the chromatin fractions, possibilities. Preliminary data suggest that was very rapidly degraded (figs 1 and 2). the differential sensitivity of the chromatin Exptl Cell Res 92 (1975)
Preliminary notes
52 1
Fig. 2. Abscissa: time (min); ordinate: % DNA digested. O---O, condensed chromatin; A-*-A, intermediate chromatin; q . . 0, extended chromatin I; O-0, extended chromatin II. DNA isolated from condensed chromatin (0-0); intermediate chromatin (A-A); extended chromatin I (m-m); and extended chromatin II (+-+I. Rate of digestion of chromatin fractions isolated from Chinese hamster liver. Chromatin fractions or purified DNA samples were adjusted to contain 25 pg/ml equivalent of DNA, and the assays were performed with pancreatic DNase I at 5 pg/ml. Each point represents the average of two determinations.
to DNase is a result of differences in presumptive euchromatin and heteroin the binding of proteins to DNA rather chromatin has several interesting implications. The relatively greater inaccessibility of the than the degree of condensation of the DNA in heterochromatin, compared to chromatin. Simpson & Polacow [24] used euchromatin, may possibly explain the tranmicrococcal nuclease to study DNA-protein interactions in condensed and extended scriptional inactivity of this condensed chrochromatin fractions obtained from calf matin, for the RNA polymerase may be blocked from reaching the promotor site, or thymus and rabbit liver by ECTHAMcellulose chromatography. Their work also prevented from transcribing the DNA. In indicates that the chromosomal proteins are this regard, Simpson has recently shown more tightly complexed to DNA in con- that there are far fewer binding sites for densed than in extended chromatin. E. coli RNA polymerase in condensed than On the basis of morphology and the in in extended chromatin [25]. vitro template activity for RNA synthesis, The difference in DNA-protein interthe condensed and extended chromatin actions between euchromatin and heterofractions obtained by the present isolation chromatin could also be an important factor procedure probably correspond to hetero- underlying the production of bands on metachromatin and euchromatin, respectively phase chromosomes. Several studies have (unpublished data; also [ll, 161). If this shown that the C-banding methods, which assumption is correct, the finding that there reveal constitutive heterochromatin, prefare differences in DNA-protein interactions erentially extract non C-band chromatin
fractions
Exptl Cell Res 92 (1975)
522
Preliminary notes
and DNA [3, 9, 211.The constitutive heterochromatin remains relatively intact during the induction of C-bands, and is clearly resistant to extraction. G-bands may represent intercalary heterochromatin [8], and electron microscopy of G-banded chromosomes, produced by trypsinization, has shown that the G-bands are areas of packed chromatin fibres which are relatively more resistant to dispersion than the interbands [2, 31. The increased inaccessibility of DNA in heterochromatin, demonstrated by DNase digestion, could readily account for the resistance of the DNA in constitutive heterochromatin to extraction by C-banding methods and the relatively greater resistance of G-band regions to dispersion during trypsinization, compared to interbands. Recent experiments involving the digestion of metaphase chromosomes with DNase suggest that the DNA in the band regions is, in fact, less accessible to DNase than that in the interbands [4]. The differential accessibility of the DNA in the band and interband regions is probably due to differences in DNA-protein interactions. Supported by Grant MA-5125 from the Medical Research Council of Canada. G.D.B. is a Medical Research Council Scholar.
Exptr Cell Res 92 (197.5)
References 1. Billing, R J & Bonner, J, Biochim biophys acta 281 (1972) 453. 2. Burkholder, G D, Nature 247 (1974) 292. 3. - Exp cell res 90 (1975) 269. 4. - In preparation. 5. Burton, K, Biochem j 62 (1956) 315. 6. Chauveau, J, Moule, Y & Rouiller, C H, Exp cell res11 (1956) 317. 7. Clark, R J & Felsenfeld, G, Nature new biol 229 (1971) 101. 8. Comings, D E, Adv human genet 3 (1972) 237. 9. Comings, D E, Avelino, E, Okada, T A & Wyandt, H E, Exp cell res 77 (1973) 469. 10. Comings, D E. Personal communication. 11. Frenster, J H, Allfrey, V G & Mirsky, A E, Proc natl acad sci US 50 (1963) 1026. 12. Itzhaki, R F, Biochem j 125 (1971) 221. 13. - Eur j biochem 47 (1974) 27. 14. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 15. Marmur, J, J mol biol 3 (1961) 208. 16. Mattoccia, E & Comings, D E, Nature new biol 229 (1971) 175. 17. Mirsky, A E, Proc natl acad sci US 68 (1971) 2945. 18. Mirsky, A E & Silverman, B, Proc natl acad sci US 69 (1972) 2115. 19. Mirsky, A E, Silverman, B & Panda, N C, Proc natl acad sci US 69 (1972) 3243, 20. Murphy, E C, Hall, S H, Shepherd, J H & Weiser, R S, Biochemistry 12 (1973) 3843. 21. Pathak, S & Arrighi, F E, Cytogenet cell genet 12 (1973) 414. 22. Pederson, T, Proc natl acad sci US 69 (1972) 2224. 23. Reeck, G R, Simpson, R T & Sober, H A, Proc natl acad sci US 69 (1972) 2317. 24. Simpson, R T & Polacow, I, Biochem biophys res commun 55 (1973) 1078. 25. Simpson, R T, Proc natl acad sci US 71 (1974) 2740. Received February 26, 1975.
erythrocyte heterokaryons [8, 181.Although 16. Ege, T, Carlsson, S-A & Ringertz, N R, Exptl cell res 69 (1971) 472. the accumulation of T antigen in chick 17. Dupuy-Coin, A-M, Ege, T, Bouteille, M & Ringertz, N R. In preparation. erythrocyte nuclei closely paralleled nuclear 18. Appels, R, Tallroth, E, Appels, D M & Ringertz, enlargement this is partly coincidental beN R, Exptl cell res 92 (1975) 70. causeprevious results obtained by Koprowski 19. Koprowski, H, Jensen, F C & Steplewski, Z, Proc natl acad sci US 58 (1967) 127. et al. [19,20] show that the T antigen can also 20. Steplewski. Z. Knowles. B & Konrowski. H. ’ ’ Proc natl acad sci US 59’(1968) 769.‘ migrate into non-transformed nuclei which are already active before fusion. The migra- Received February 17, 1975 tion of T antigen into reactivating chick erythrocyte nuclei is of interest not only as a part of the reactivation process but also because chick cells cannot be transformed or Differential accessibility of DNA in extended infected with SV40. Nuclear uptake of SV40 and condensedclwomatin to pancreatic DNase I specified T antigen therefore is independent and M. G. WEAVER, of the permissivenessand transformability of G. D. BURKHOLDER Department of Anatomy, College of Medicine, Unithe mammalian cell. versity of Saskatchewan, Saskatoon, Saskatchewan S7N 0 WO, Canada This investigation was supported by grants from the Swedish Cancer Society and Sigrid Juselius Foundation. The authors wish to thank Dr J. van der Noordaa, University of Amsterdam, Holland, for the generous gift of T antisera.
References 1. Cameron, I & Prescott, D, Exptl cell res 56 (1969) 406. 2. Ringertz, N R & Bolund, L. The cell nucleus (ed H Busch) vol. III, p. .417. Academic Press, New York (1974). Harris, H, J cell sci 2 (1967) 23. Nucleus and cytoplasm. Oxford Univ Press, oxford, UK (1968). Bolund, L, Ringer& N R & Harris, H, J cell sci 4 (1969) 71. Ringer& N R & Bolund, L, Int rev exp path01 (ed G W Richter & M A Epstein) vol. 13, p. 83. Academic Press, New York (1974). 7. Ringertz N R, Carlsson, S-A Ege, T & Bolund, L, Proc natl acad sci US 68 (1971) 3228. 8. Appels, R, Bolund, L & Ringertz N R, J mol biol 87 (1974) 339. 9. Appels, R & Ringertz N R, Current topics of dev biol (ed A MONOY & M Moscona). Academic Press, New York (1975). In press. Naha, P M. Exntl cell res 80 (1973) 467. :?I Butel, J S, Terethia S S & Melnick, J L, Adv in cant res (ed G Klein) vol. 15, p. 55. Academic Press, New York (1972). 12. Girardi A J, Jensen, F C & Koprowski, H, J cell camp physiol 65 (1965) 69. 13. Carlsson, S-A, Savage, R E & Ringertz N R, Nature 228 (1970) 869. 14. Harris. H. Watkins. J F. Ford. C E & Schoefl. G I, J’cellsci 1(1966) 1.. 15. Stenman, S, Zeuthen, J & Ringertz, N R, Int j cancer res (1975). 15 (1975) 547. Exptl CeN Res 92 (1975)
Summary. Pancreatic DNase I has been used to study the interaction between DNA and chromosomal proteins in extended and condensed chromatin fractions isolated from mouse and Chinese hamster livers. It was found that DNase digests extended chromatin at a faster rate than condensed chromatin, and the evidence suggests that the chromosod proteins are more tightly complexed to the DNA in condensed than in -extended -chromatin. This difference in DNA-protein interaction in extended and condensed cbromatin may be related to the functional difference which characterizes these fractions, and might be one of the factors underlying the production of bands on metaphase chromosomes.
Deoxyribonuclease (DNase) has served as a useful probe for investigating the structural properties of chromatin [l, 7, 12, 13, 17-19, 221. Purified DNA is rapidly degraded by DNase, however the DNA in chromatin is afforded a limited measure of protection against nuclease attack, primarily as a result of the association of chromosomal proteins with the DNA [17, 221.The rate of digestion of chromatin DNA in the presence of pancreatic DNase I is dependent on the degree of binding of proteins to the DNA [22]. Chromatin in which the DNA is loosely associated with protein will be digested at a faster rate than chromatin in which the DNA is more tightly associatedwith protein.
Preliminary notes
It is therefore possible to study relative rates of digestion as a means to distinguish relative differences in chromatin structure. Several methods currently exist for separating chromatin into presumptive euchromatin (extended) and heterochromatin (condensed) fractions [II, 16, 20, 231. We have used one of these methods to obtain extended and condensed fractions from mouse and Chinese hamster livers, and have assayed the degree of complexing between DNA and chromosomal proteins in these fractions using pancreatic DNase I. Materials and Methods Liver nuclei were isolated from Chinese hamsters (Cricetulus griseus) or CsH mice by the method of Chauveau et al. [6], as modified by Comings [lo]. The livers of several fasted animals were perfused with ice-cold 0.01 M Tris buffer, pH 8.0, containing 3 x 1O-JM MgC12, 1O-8 M NaHS03, and 1O-4M CdSOd. Subsequent procedures were performed at 4°C. After mincing, the livers were homogenized in 2.4 M sucrose, 3.3 x 1O-8 M CaCIZ, and 1O-8 M NaHSO, (SCN), using a teflon homogenizer. When most of the cells were broken, the homogenate was filtered through cheesecloth and layered on top of a 4th tube volume of SCN. The interface between the two layers was mixed to form a crude gradient, and the tubes were centrifuged at 40080 g for 1 h. The nuclear pellet was subsequently washed according to the following schedule: 1 x in 0.01 M Tris buffer, pH 7.0, 5 x 1O-s M MgCl,, 3 x 1O-s M CaCl,, lo-* M NaHS03, 1O-4 M CdSOI, 0.14 M NaCl, 1 pg/ml soybean trypsin inhibitor (SW buffer) + lo-* M phenylmethylsulfonylfluoride (Calbiochem); ’ 3 x in SW buffer alone; 1 x in SW buffer + 0.05 % Triton X-100; 1 x in SW buffer alone. The nuclei were pelleted at 400 g for 5 min after each wash. The isolation of chromatin subfractions was a modification of the method of Comings [lo, 161. Washed nuclei were resuspended in 0.34 M sucrose, 0.1 pg/ml soybean trypsin inhibitor, 0.01 M Tris buffer, pH 7.0, and were left on ice for 15 min. The nuclear suspension was then sonicated with a Branson W-140 sonicator (standard horn; setting no. 8), using 10 set bursts, until phase contrast microscopy indicated that the majority of the nuclei were ruptured. Centrifugation at 400 g for 5 min pelleted unbroken nuclei, and chromatin fractions were obtained from the supernatant by differential centrifugation. The pellet obtained by centrifugation at 4000 g for 20 min contained the condensed chromatin fraction. The supematant was then centrifuged at 20 000 g for 20 min to yield the intermediate chromatin fraction, and the supernatant was recentrifuged at 100000 g for 30 min. The resulting pellet was taken as the extended chromatin fraction I, and the supematant was
5 19
the extended chromatin fraction 11. All fractions were dialyzed overnight against 0.01 M Tris buffer, nH 7.0. The purity of the chromatin fractions was determined on a morphological basis, using electron microscopy. The condensed fraction also contained nucleoli, and appeared slightly contaminated by extended chromatin. The extended chromatin fractions were extremely pure, containing little or no condensed chromatin. The intermediate fraction was a mixture of condensed and extended chromatin. DNA and protein estimations were determined using the Burton [5] and Lowry [14] assays, respectively. The protein/DNA ratios of the mouse chromatin fractions (mean of 5 determinations) were as follows: condensed, 2.36; intermediate, 2.26; extended I, 1.91; extended II, 1.65. Each dialyzed chromatin fraction was adjusted with 0.01 M Tris buffer, pH 7.0, to contain 25 pig/ml equivalent of DNA, as determined by the absorbance at 260 nm. The chromatin fractions were then warmed to 37°C and MgC& and bovine pancreatic DNase 1 (Worthington) were added in sequence to final concentrations of 3 x lO-s M and 5 pg/ml, respectively. The fractions were incubated in a shaker bath (37”C), and 1 ml samples were removed at various times thereafter. The reaction was stopped and undigested chromatin in each sample was precipitated by the addition of ice-cold perchloric acid to a final concentration of 0.25 M. The samples were left on ice for 30 min and then centrifuged at 37 000 g for 15 min to pellet undigested chromatin. The amount of digested DNA in each supernatant was quantitated by measurement of the absorbance at 260 nm. As a control, DNA was isolated from each of the chromatin fractions using the method of Marmur [15]. This purified DNA was exposed to DNase I using the same procedure as for the chromatin samples.
Results
The percentage of DNA digested in the chromatin and purified DNA fractions exposed to DNase was plotted as a function of time to give the rate of digestion (figs 1 and 2). All of the chromatin fractions exhibited two steps in their digestion rate: a fast initial rate, with 40-65 % of the DNA being digested during the first 15 min, and a slower rate of digestion thereafter. Of significance was the finding that the rates of digestion differed in the various chromatin fractions. Extended chromatin was digested at a faster rate than condensed chromatin in both the mouse and Chinese hamster (figs 1 and 2), so that, at all sampling times, a higher Exptl Cell Res 92 (1975)
520 Preliminary notes
These DNA fractions were digested at a uniform rate, and there were no differences in digestion rates comparable to their chromatin counterparts. ‘Discussion
In both the mouse and Chinese hamster, DNA in extended chromatin was more sensitive to the action of DNase than that in condensed chromatin, and the intermediate chromatin fraction generally appeared to have an intermediate sensitivity (figs I and 2). This differential sensitivity of the chromatin fractions to DNase digestion was not inherent in the DNA each contained, since Fig. I. Abscissa: time (min); ordinate: % DNA DNA samples purified from the chromatin Condensed chromatin fraction; digested. O---O, fractions did not differ in their rate of n-.-A, intermediate chromatin fraction, 0. * 0, extended chromatin fraction I. DNA isolated from digestion. The difference in the rates of condensed chromatin (O-O); intermediate chrodigestion of the chromatin fractions must matin fraction 1 (H-W). Rate of digestion of chromatin fractions isolated therefore be related to the association of the from mouse liver. Chromatin fractions or Durified chromosomal proteins with the DNA, such DNA samples were adjusted to contain 25 pg/ml e$uivalent of DNA, and the assays were performed that the DNA in condensed chromatin is with pancreatic DNase I at 5 pg/ml. See text for protected from DNase attack to a greater details. extent than that in extended chromatin. There appear to be two possible ways in percentage of DNA was digested in extended which this protection might be achieved: compared to condensed chromatin. Eight (1) The DNA in condensed chromatin may separate experiments have been conducted be more resistant to the action of DNase with isolated mouse chromatin fractions, simply becausethis chromatin is more folded and three with Chinese hamster fractions, all than the extended chromatin. It is easy to with the same result. envisage that DNA would be less accessible, The intermediate chromatin fraction was to DNase, within a mass of tightly folded somewhat variable in its rate of digestion, chromatin than in relatively unfolded chroranging from a rate similar to that of con- matin. (2) The chromosomal proteins may densed chromatin (fig. 1) to an intermediate be more tightly complexed to the DNA in condensed compared to extended chromatin. rate of digestion (fig. 2). This variability presumably reflects the degree of inter- As a result, the DNA in condensedchromatin mixing of condensed and extended chromatin would be more inaccessible to DNase than that in extended chromatin, and would in this fraction. Purified DNA, isolated from the extended, therefore be afforded a greater measure of intermediate, and condensed chromatin frac- resistance to DNase attack. Additional work tions, and exposed to DNase using the same is in progress to distinguish between these conditions as for the chromatin fractions, possibilities. Preliminary data suggest that was very rapidly degraded (figs 1 and 2). the differential sensitivity of the chromatin Exptl Cell Res 92 (1975)
Preliminary notes
52 1
Fig. 2. Abscissa: time (min); ordinate: % DNA digested. O---O, condensed chromatin; A-*-A, intermediate chromatin; q . . 0, extended chromatin I; O-0, extended chromatin II. DNA isolated from condensed chromatin (0-0); intermediate chromatin (A-A); extended chromatin I (m-m); and extended chromatin II (+-+I. Rate of digestion of chromatin fractions isolated from Chinese hamster liver. Chromatin fractions or purified DNA samples were adjusted to contain 25 pg/ml equivalent of DNA, and the assays were performed with pancreatic DNase I at 5 pg/ml. Each point represents the average of two determinations.
to DNase is a result of differences in presumptive euchromatin and heteroin the binding of proteins to DNA rather chromatin has several interesting implications. The relatively greater inaccessibility of the than the degree of condensation of the DNA in heterochromatin, compared to chromatin. Simpson & Polacow [24] used euchromatin, may possibly explain the tranmicrococcal nuclease to study DNA-protein interactions in condensed and extended scriptional inactivity of this condensed chrochromatin fractions obtained from calf matin, for the RNA polymerase may be blocked from reaching the promotor site, or thymus and rabbit liver by ECTHAMcellulose chromatography. Their work also prevented from transcribing the DNA. In indicates that the chromosomal proteins are this regard, Simpson has recently shown more tightly complexed to DNA in con- that there are far fewer binding sites for densed than in extended chromatin. E. coli RNA polymerase in condensed than On the basis of morphology and the in in extended chromatin [25]. vitro template activity for RNA synthesis, The difference in DNA-protein interthe condensed and extended chromatin actions between euchromatin and heterofractions obtained by the present isolation chromatin could also be an important factor procedure probably correspond to hetero- underlying the production of bands on metachromatin and euchromatin, respectively phase chromosomes. Several studies have (unpublished data; also [ll, 161). If this shown that the C-banding methods, which assumption is correct, the finding that there reveal constitutive heterochromatin, prefare differences in DNA-protein interactions erentially extract non C-band chromatin
fractions
Exptl Cell Res 92 (1975)
522
Preliminary notes
and DNA [3, 9, 211.The constitutive heterochromatin remains relatively intact during the induction of C-bands, and is clearly resistant to extraction. G-bands may represent intercalary heterochromatin [8], and electron microscopy of G-banded chromosomes, produced by trypsinization, has shown that the G-bands are areas of packed chromatin fibres which are relatively more resistant to dispersion than the interbands [2, 31. The increased inaccessibility of DNA in heterochromatin, demonstrated by DNase digestion, could readily account for the resistance of the DNA in constitutive heterochromatin to extraction by C-banding methods and the relatively greater resistance of G-band regions to dispersion during trypsinization, compared to interbands. Recent experiments involving the digestion of metaphase chromosomes with DNase suggest that the DNA in the band regions is, in fact, less accessible to DNase than that in the interbands [4]. The differential accessibility of the DNA in the band and interband regions is probably due to differences in DNA-protein interactions. Supported by Grant MA-5125 from the Medical Research Council of Canada. G.D.B. is a Medical Research Council Scholar.
Exptr Cell Res 92 (197.5)
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