John H. Frenster, Vincent G. Allfrey, and Alfred E. Mirsky

Rockefeller Institute, New York, N.Y. 10021

Communicated October 22, 1963 

Up to 80 percent of the deoxyribonucleic acid (DNA) of interphase thymus lymphocytes is inactive in messenger ribonucleic acid (RNA) synthesis (1), and is apparently repressed (2), correlating well with the differentiated state of these cells (3). It has long been suggested that inactive chromosomal segments could be recognized cytologically during interphase and early prophase by their dense staining characteristics, implying a condensed state (see ref. 4 for general review). Such condensed inactive chromosomal segments have been termed heterochromatin (5-7), in contrast to segments in a more active, extended, diffusely staining state, termed euchromatin.

The heterochromatic sex-chromatin body of Barr (8) in female mammalian cells is composed of a segment of one X chromosome (9-11), and the genes carried on this heterochromatic segment have been shown to be unexpressed in the phenotype of the cell (12-14). The DNA of this heterochromatic X chromosome replicates later than that of other chromosomal segments (15-17), and it has been shown that heterochromatic segments of interphase autosomes are much less active in RNA synthesis (6) and in DNA synthesis (18) than are euchromatic segments.

Much of the DNA of interphase thymus lymphocytes is visible cytologically as condensed, Feulgen-positive masses (19, 20), resembling the heterochromatin of the inactive Barr sex-chromatin body. In this study it is shown that these condensed chromosomal segments can be isolated in mass from lymphocytes, that up to 80 percent of the DNA of interphase lymphocytes is contained in these masses, and that the DNA, RNA, and protein metabolism of these condensed heterochromatin masses is decreased compared to that of the more-extended euchromatin fibrils. 

Previous isolations of chromatin:

Chromatin represents interphase chromosomal segments and, as such, contains DNA, histones, nonhistone proteins, RNA, lipids, and perhaps polysaccharides (21). The native association of such components is disrupted by marked departures of the isolation medium from isotonicity (salt concentrations above 0.6 M or below 0.01 M) or from neutrality (above pH 8.0 or below pH 6.0) (22), imposing constraints upon the isolation procedure (23). Previous methods of isolation of chromatin have utilized blending (24, 25), grinding with sand (26) or with glass beads (27), or ultrasonication (28, 29) of isolated nuclei that have been washed free of cytoplasm. In the method reported here, nuclear membranes, nuclear ribosomes, and soluble nuclear proteins were also removed before the isolation of chromatin fractions, and gelation was prevented throughout.

Isolation of interphase nuclei:

A slight modification of the methods previously described (30) was used. Calf thymus lobes were brought to the laboratory in an ice-cold solution of 0.25 M sucrose within 1 hr of slaughtering. All subsequent operations other than incubation were performed at 2^o C. The thymus of a single animal was selected, dissected free of adherent fat, connective tissue, and blood, and 100 gm of wet-weight tissue was finely minced. The mince was suspended in 1000 ml of 0.25 M sucrose-0.0033 M CaCl₂, and was homogenized at half speed in a Waring blendor 3 min. After filtering through cheesecloth, the homogenate was blended twice more for 3 min, with filtrations through flannelette, and was then centrifuged at 600 g for 7 min. The resultant nuclear pellet was washed once 1000ml of 0.25 M sucrose-0.0033 M CaCl₂, and was then suspended in 100 ml of an incubation medium of composition: 0.1875 M sucrose, 0.02 M glucose, 0.025 M Tris HCl, 0.0128 M NaCl, 0.0033 M CaCl₂, pH 7.1.

Incubation of nuclei:

C¹⁴-labelled precursors, such as thymidine-2-C¹⁴, uridine-2-C¹⁴, orotic acid-6-C¹⁴, alanine-1-C¹⁴, and lysine-1-C¹⁴ were added to the incubation suspensions, as shown in the legends to the tables, and were incubated at 37^o C for 30 min in air with agitation. The incubation suspension was then added to 900 ml of cold incubation medium, and centrifuged at 600 g for 7 min.

Isolation of subnuclear fractions:

The supernatant incubation medium contained cytoplasmic fragments and nuclear membranes (31), which were removed in this and a second washing in 1000 ml of incubation medium. The washed nuclear pellet was extracted three times with 1000-ml volumes 0.01 M Tris HCl-0.0033 M CaCl₂, pH 7.1, removing nuclear ribosomes and neutral proteins (31). The easily decantable slurry following the third Tris extraction was removed, and the residual extracted nuclei were resuspended in 1000 ml of cation-free 0.25 M sucrose, allowed to stand for 10 min to permit swelling of the nuclei, and were then sonicated for 5 sec at 20,000 cycles per second and 7 amp on a Branson S-75 sonifier. Such brief sonication is much less than the 7-20 min employed in previous isolations of chromatin (28, 29). Previous studies of the effect of sonication on isolated DNA showed that 5 sec exposure had minimal effects on the physical (32) and biological (33) properties of protein-free DNA, and that the native association of histone with DNA, as in chromatin, further stabilizes DNA against physical change (22).

The suspension was sonicated in aliquotes of 60 ml at a time, and the resulting sonicate was centrifuged at 100 g for 5 min, with the supernate being filtered through four layers of flannelette to remove any aggregated material. This filtrate was centrifuged at 1000 g for 10 min, and the pellet collected as condensed (hetero-)chromatin masses. The supernate was then centrifuged at 3000 g for 30 min, and the pellet collected as the intermediate fraction. The supernate obtained was then centrifuged at 78,000 g for 60 min, and this pellet collected as extended (eu-)chromatin fibrils. The resultant clear supernate was then made 10 mM in calcium chloride and centrifuged at 1000 g for 10 min. This pellet was collected as particles in 10 mM calcium. The measurement of protein, DNA, RNA, and radioactivity was as previously described (31).

Electron microscopy:

Electron micrographs of the whole tissue mince before the addition of solutions, and of the various centrifuge pellets, were obtained after fixation of the tissues for 15 min in 1% OsO₄ (20), dehydration in alcohol, embedding in Epon (34), sectioning at 800-A thickness on a Cambridge-Huxley microtome, and staining for 15 min in 1% uranyl acetate (20). Such staining is needed to reveal fully the condensed (hetero-)chromatin masses as seen by phase contrast or ultraviolet microscopy (35, 36). A Phillips model EM-100 A electron microscope was used, yielding microscopic magnifications to X 20,000. Subsequent magifications were obtained photographically.

Electron micrographs of thymus tissue sampled before the addition of preparative solutions (Figs. 1 and @) reveal large masses of condensed (hetero-)chromatin arrayed peripherally toward the margins of the lymphocyte nucleus, with broad-based attachments to the nuclear membrane. These masses are seen at higher maginifications to be composed of a dense reticulum of 100-A fibrils, and are similar to those reported previously in thymus lymphocytes (3, 19, 20), nucleated erythrocytes (37), blood lymphocytes (38), pancreas (20, 39), liver (40), von Kupfer cells and granulocytes (41), lymphosarcoma cells (36), Ehrich ascites cells (42), and as karyosomes (43) or chromocenters (5) in other well-differentiated cells. Fine strands of extended 50-A (eu-)chromatin fibrils are seen coursing between the large condensed masses, and an occasional light-staining 0.5-u nucleolus is seen, usually at the border of a condensed chromatin mass (20).

Electron micrographs of isolated nuclei following extraction of nuclear ribosomes and neutral proteins (Figs. 3 and 4) reveal these same features, but when the extracted nuclei are resuspended in cation-free isotonic sucrose (Figs. 5 and 6), the nuclei swell to twice their normal size, the extended chromatin fibrils are spread, and these are seen to course from the periphery of the condensed chromatin masses (44, 45). Following brief sonication and isolation of the condensed (hetero-)chromatin masses (Figs. 7 and 8), their basic structure and dimensions remain unaltered, with some fragmented euchromatin fibrils still attached. Isolated euchromatin fibrils (Fig. 9) are seen to be free of condensed chromatin masses. The isolated particles in 10 mM calcium (Fig. 10) appear as aggregates of particles somewhat resembling those of isolated nucleoli (29) or their fragments. The intermediate fraction appears as a mixture of condensed (hetero-)chromatin masses and extended (eu-)chromatin fibrils.

The metabolism of DNA, RNA, and protein of the condensed chromatin masses and of the extended chromatin fibrils was studied by means of incubations of the nuclei with C¹⁴-labelled precursors. Over 80 percent of the protein, DNA, RNA, and counts incorporated into new RNA were recovered among the subnuclear fractions (Table 1).
 

TABLE 1. Incorporation of Orotic Acid-6-C¹⁴ into RNA of Subnuclear Fractions*

Fraction	Protein (mg)	DNA (mg)	RNA (mg)	RNA, total counts (cts/min)	RNA,  specific activity (cts/min/mg)	Composition DNA / RNA
Whole nucleus	1505.2	493.0	48.40	717,000	14,400	10.2
Cytoplasmic fragments and nuclear membranes	57.5	1.59	2.26	5,085	2,250	0.70
Nuclear ribosomes and  pH 7.1-extracted proteins	253.6	14.37	24.73	38,200	1,543	0.58
Loose slurry after pH 7.1 Tris extraction	129.1	48.40	3.06	101,000	33,000	15.8
Condensed  (hetero-)chromatin	481.5	259.26	7.14	71,855	10,600	36.3
Intermediate	136.8	47.39	3.10	63,230	20,400	15.3
Extended (eu-)chromatin	117.2	44.60	6.23	207,600	33,300	7.1
Particles in 10 mM calcium	65.9	8.95	1.75	106,000	60,600	5.1
Recovery	1237.6	424.44	48.27	592,970
Percent	82	86	97	83

*Orotic acid-6-C¹⁴ (5.0 mg, 1.86 mC) added to suspension of isolated nuclei in 100 ml of incubation medium (see Materials and Methods). 

^*Thymidine-2-C¹⁴ incorporated into DNA, after incubation of nuclei with 0.47 mg (50 uC) in 100 ml of incubation medium (see Materials and Methods).
⁺Uridine-2-C¹⁴ incorporated into RNA, after incubation of nuclei with 0.06 mg (7.5 uC) in 100 ml of incubation medium (see Materials and Methods). 

*dl-Alanine-1-C¹⁴ incorporated into protein after incubation of nuclei with 3.72 mg (169 uC) of isotope in 100 ml of incubation medium (see Materials and Methods).
⁺dl-Lysine-1-C¹⁴ incorporated into protein after incubation of nuclei with 5.0 mg (98 uC) of isotope in 100 ml of incubation medium (see Materials and Methods). 

The gene-inactivation hypothesis of cell differentiation postulates that as a cell differentiates, those genes not mediating the particular state of differentiation are inactivated by a repression mechanism (1, 49-52). The present study confirms previous evidence (1, 2) that a large fraction of the DNA of interphase calf thymus lymphocytes is metabolically inactive, correlating well with the differentiated state of these cells (3).

The isolation of repressed and active chromatin from interphase lymphocytes permits studies to elucidate the mechanism of repression in condensed (hetero)-chromatin. If this isolation procedure is gentle enough, the repressed and active chromatin fractions may demonstrate the native in vivo association of DNA, histones, nonhistone proteins, RNA, lipids, and other molecular species, permitting an assessment of their role in repression. The histone hypothesis of DNA inactivation is a plausible mechanism for the maintainence of chromatin repression (1, 2, 25, 53). Current studies in progress (54) suggest that the active, extended (eu-)chromatin fibrils, as isolated by this procedure, have a deficiency of histone for their DNA content, as contrasted to the balance between histone and DNA in the inactive, condensed (hetero-)chromatin masses. A partial account of this study has been presented (55). 

Repressed and active chromatin can be isolated in mass from interphase calf thymus lymphocytes. Up to 80 percent of interphase lymphocyte DNA is found in the repressed chromatin fraction. Repressed interphase DNA appears as condensed (hetero-)chromatin, while active interphase DNA appears as extended (eu-)chromatin fibrils. The extended (eu-)chromatin fraction contains the bulk of the rapidly labeled RNA of the nucleus. 

2. Allfrey VG, Littau VC, and Mirsky AE, these Proceedings 49, 414 (1963).

3. Sainte-Marie G, and Leblond CP, Proc. Soc. Exp. Biol. Med. 98, 909 (1958).

4. Cooper KW, Chromosoma 10, 535 (1959).

5. Heitz E, Ber. Deut. Botan. Ges. 47, 274 (1929).

6. Hsu TC, Exptl. Cell Res. 27, 332 (1962).

7. Grumbach MM, Morishima A, and Taylor JH, these Proceedings 49, 581 (1963).

8. Barr ML, and Bertram EG, Nature 163, 676 (1949).

9. Ohno S, Kaplan WD, and Kinosita R, Exptl. Cell Res. 18, 415 (1959).

10.Ohno S, and Hauschka TS, Cancer Res. 20, 541 (1960).

11. Ohno S, and Makino S, Lancet 1, 78 (1961).

12. Lyon M, Nature 190, 372 (1961).

13. Russell LB, Science 133, 1795 (1961).

14. Beutler E, Yeh M, and Fairbanks UF, these Proceedings 48, 9 (1962).

15. Lima-de-Faria A, J. Biophys. Biochem. Cytol. 6, 457 (1959).

16. Taylor JH, J. Biophys. Biochem. Cytol. 7, 455 (1960).

17. German JL III, Trans. N.Y. Acad. Sci. 24, 395 (1962).

18. Hay ED, and Revel JP, J. Cell Biol. 16, 29 (1963).

19. Ris H, and Mirsky AE, J. Gen. Physiol. 32, 489 (1949).

20. Huxley HE, and Zubay G, J. Biophys. Biochem. Cytol. 11, 273 (1961).

21. Mirsky AE, and Pollister AW, J. Gen. Physiol. 30, 117 (1946).

22. Zubay G, and Doty P, J. Mol. Biol. 1, 1 (1959).

23. Frenster JH, Exptl. Cell Res. Suppl. 9, 235 (1963).

24. Mirsky AE, and Pollister AW, Trans. N.Y. Acad. Sci. 5, 190 (1943).

25. Huang R, and Bonner J, these Proceedings 48, 1216 (1962).

26. Claude A, and Potter JS, J. Exp. Med. 77, 345 (1943).

27. Birnstiel ML, and Hyde BB, J. Cell Biol. 18, 41 (1963).

28. Rees KR, Rowland GF, and Varcoe JS, Biochem. J. 86, 130 (1963).

29. Maggio R, Siekevitz P, and Palade GE, J. Cell Biol. 18, 293 (1963).

30. Allfrey VG, Mirsky AE, and Osawa S, J. Gen. Physiol. 40, 451 (1957).

31. Frenster JH, Allfrey VG, and Mirsky AE, these Proceedings, 46, 432 (1960).

32. Doty P, McGill BB, and Rice SA, these Proceedings 44, 432 (1958).

33. Litt M, Marmur J, Ephrussi-Taylor H, and Doty P, these Proceedings 44, 144 (1958).

34. Luft JH, J. Biophys. Biochem. Cytol. 9, 409 (1961).

35. Gibbons IR, and Bradfield JRG, Biochim. et Biophys. Acta 22, 506 (1956).

36. Bernhard W, and Granboulan N, Expt. Cell Res. Suppl. 9, 19 (1963).

37. Davies HG, and Spencer M, J. Cell Biol. 14, 445 (1962).

38. Lima-de-Faria L, and Reitalu J, J. Cell Biol. 16, 315 (1963).

39. Leduc EH, and Bernhard W, J. Biophys. Biochem. Cytol. 10, 437 (1961).

40. Watson ML, and Aldridge WG, J. Biophys. Biochem. Cytol. 11, 257 (1961).

41. Maggio R, Siekevitz P, and Palade GE, J. Cell Biol. 18, 267 (1963).

42. Swift H, Exptl. Cell Res. Suppl. 9, 54 (1963).

43. Wilson EB, The Cell in Development and Heredity (New York: MacMillan Co., 1928), 3rd ed., p. 93.

44. Crawley JCW, and Harris H, Exptl. Cell Res. 31, 70 (1963).

45. Smetana K, Steele WJ, and Busch H, Exptl. Cell Res. 31, 198 ( 1963).

46. Edstrom JE, and Beermann W, J. Cell Biol. 14, 371 (1962).

47. Gall JG, and Callan HG, these Proceedings 48, 662 (1962).

48. Izawa M, Allfrey VG, and Mirsky AE, these Proceedings 49, 544 (1963).

49. Mather K, Nature 161, 872 (1948).

50. Stedman E, and Stedman E, Nature 166, 780 (1950).

51. McClintock B, in Genetic Mechanisms: Structure and Function, Cold Spring Harbor Symposia on Quantitative Biology, vol. 21 (1956), p. 197.

52. Beermann W, in Genetic Mechanisms: Structure and Function, Cold Spring Harbor Symposia on Quantitative Biology, vol. 21, (1956), p. 217.

53. Frenster JH, Allfrey VG, and Mirsky AE, Biochim. et Biophys. Acta 47, 130 (1961).

54. Allfrey VG, Frenster JH, and Mirsky AE, in press.

55. Frenster JH, J. Cell Biol. 19, 25A (1963). 

1. Electron Microscopy of Human Lymphocytes before and after Activation by PHA (Busch H, 1974).

2. Frenster JH,"Ultrastructural Continuity between Active and Repressed Chromatin",  Nature 205: 1341 (1965).

3. Frenster JH, "Nuclear Polyanions as De-Repressors of Synthesis of Ribonucleic Acid", Nature 206: 680 (1965).

4. Frenster JH, "Mechanisms of Repression and De-Repression within Interphase Chromatin", In-Vitro 1:
pp. 78-101 (1965).