Soluble nucleohistone of compact configuration

Biochimica et Biophysica Acta, 361 (1974) 97--108 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98053 SOLUBLE NUCLEOHISTONE OF COMPACT CONFIGURATION

ALLAN W. REES, MICHAEL S, DEBUYSERE and EDWIN A. LEWIS Department o f Biochemistry, The University o f Texas Health Science Center at San Antonio, San Antonio, Texas 78284 (U.S.A.)

(Received December 12th, 1973) (Revised version received April 1st, 1974 )

Summary Nucleohistone solubilized by an autolysis of rabbit t h y m u s nuclei, a m e t h o d avoiding shear, has an exceptionally high sedimentation velocity and low intrinsic viscosity, and is freely soluble in 0.15 M buffers. The heterogenous product was fractionated by gel exclusion chromatography. Depending on the fraction examined s~0,w was from under 70 S to over 250 S, [7] from 0.3 to 6 dl/g, molecular weight from 1 • 107 to 2 • 10 s Molecular weight of the DNA was from 6 . 1 0 6 to 2 . l 0 T and by viscosity was 8--28 times more extended than the nucleohistone particles from which it was derived. All five histones are present, with two riew bands taken to be specific cleavage products. Histone: non-histone protein: RNA: DNA is approximately 1.1 : 0.2: 0:1 by mass. Circular dichroism indicates extensive unstacking of the DNA bases. Neither repetitive nor unique DNA sequences are preferentially solubilized. The nucleohistone may be precipitated either by shearing or by dissociation--reassociation by adding salt and diluting with water, thus its solubility arises not from deproteinization but from fragile details of structure. Its shear sensitivity suggests that chromatin solubilized conventionally by shearing may thereby be altered in some structural details.

Introduction Studies of soluble chromatin by h y d r o d y n a m i c methods have shown that the nucleohistone particles contain their DNA in a configuration less extended than in purified DNA [1--8]. A supercoiled structure for nucleohistone has been proposed on the basis of X-ray diffraction [9]. A distortion of the DNA in nucleohistone has been shown by circular dichroism [8,10--15] and optical r o t a t o r y dispersion [16]. The compact state is shown most directly by comparing the intrinsic viscosity of nucleohistone with that of DNA isolated therefrom. The extent of condensation varies widely among the published reports,

98 suggesting that this property is very sensitive to the manner of preparation. It is likely that the compact states observed are relics of the architecture of chromatin in the cell nucleus. We describe here a nucleohistone which exhibits this condensed state in an extreme degree. Further, it is freely soluble in 0.15 M buffers, in which most nucleohistones have minimum solubility [1,2,6,17]. Chromatin in vivo must consist of fragile molecules of enormous size and to disperse it into particles suitable for h y d r o d y n a m i c studies must require breakage of the DNA and possibly of protein cross-links. Commonly this has been done by shearing [1--18]. Degradation of chromatin to soluble fragments without shear may retain shear sensitive structural features and yield new insight into the native structure. Nucleohistone has been solubilized without shear by an enzymatic degradation, the autolysis of rabbit t h y m u s nuclei [19]. The product is very heterogeneous [19] as are other chromatin preparations [4]. We have used gel exclusion chromatography to obtain fractions of reduced heterogeneity which we describe here in some detail. Communications describing the unfractionated nucleohistone [19], its template activity [20], electron microscopy [21], and an abstract of preliminary results [22] have appeared. Materials and Methods The nucleohistone was prepared essentially as described by Rees and Krueger [19]. Purified nuclei from t h y m u s of young rabbits were gently dispersed in 0.25 M sucrose and mixed with an equal volume of the buffer 0.04 M Tris, 0.08 M K2 SO4 (made pH 7.4 with H~ SO4 ) and with 0.04 vol. of 0.05 M MgSO4. This reaction mixture was incubated at 38°C with gentle shaking. The solubilization reaction was halted by the addition of either EDTA or citrate. The unsolubilized nucleoprotein was pelleted by centrifugation at 12 000 rev./min for 10 min. The solubilized nucleohistone was chromatographed and examined, unless otherwise stated, in a buffer which is 0.1 M KC1, 0.02 M Tris, 0.005 M citric acid, made pH 7.4 with HC1, ionic strength 0.15 M. About 20 ml of the reaction mixture supernatant was applied to a 2.5 cm × 95 cm column packed with Pharmacia Sepharose 2B and eluted at 4°C with upward flow at a rate of 3.5 ml/h. Fractions of 7 ml were collected. DNA of suspensions of nuclei and reaction mixture supernatants was determined by the m e t h o d of Burton [23]. DNA of nucleohistone fractions from chromatography was done by the Burton reaction, or by A260 nm using an extinction coefficient of 24 cm 2/mg. The latter was determined from preparations on which DNA was measured both by the Burton reaction and by phosphorus assay according to Morrison [24]. Burton assays were compared to a stock solution of calf t h y m u s sodium DNA prepared according to Kay et al. [25]. Further analysis of nucleohistone required concentrating material by pelleting poo~,ed fractions. Pellets were dissolved in 1 M NaOH and assayed for DNA according to Burton [23], total protein according to Lowry et al. [26] and RNA by the orcinol reaction according to Schneider [27] with correction for reactivity of DNA. Protein assays were compared to bovine plasma albumin and RNA assays to ribose. Separate pellets of the same pools were extracted with 0.2 M H2 SO4 and histone determined by Lowry et al. [26]. Non-histone pro-

99 tein was d e t e r m i n e d on the pellet after H2 SO4 extraction, the pellet being dissolved in 1 M NaOH. Histones ext r a c t e d into 0.2 M H2 SO4 were precipitated with cold ethanol, redissolved in 0.9 M acetic acid and subjected to gel electrophoresis according to Panyim and Chalkley [ 2 8 ] . DNA was prepared for circular dichroism and renaturation rate experiments by a variant o f the m e t h o d of Huang and Bonner [ 2 9 ] . The purified DNA had a protein to DNA ratio of less than 0.03. DNA for sedimentation velocity was purified by banding in CsC1 in the preparative ultracentrifuge. Essentially 100% of the nucleohistone DNA was recovered in the band. The same molecular weights were obtained for such DNA by either sedimentation velocity or viscometry. S ed imen tation rates were measured by b o u n d a r y sedimentation in a Beckman Model E analytical ultracentrifuge with photographic ultraviolet absorption optics. To avoid shear the cells were always filled using an 18-guage needle. Convection was avoided with a 0.5% sucrose gradient form ed by diffusion (technique suggested by Dr V. Schumaker). Films were scanned using the Gilford s p e c t r o p h o t o m e t e r with film d e n s i t o m e t e r a t t a c h m e n t described by Rees [ 3 0 ] . Nucleohistone samples were run in 0.1 M KC1, 0 . 0 2 M Tris--HC1, 0.005 M citrate pH 7.4 and DNA in 0.1 M sodium phosphate, 0.01 M EDTA, pH 6.8. All results were adjusted to s: 0,w. A U of 0.665 was assumed for nucleohistone estimated from its composition. Sedimentation constants of DNA were co r r ect ed to zero c o n c e n t r a t i o n by combining the c o n c e n t r a t i o n d e p e n d en ce equation given by Eigner [31] with the equation of Crothers and Zimm [ 3 2 ] . The Crothers and Zimm equations were used to calculate molecular weights o f DNA f r om sedimentation velocities or viscosities. Viscometry was done at 20 + 0.01°C in a 4-bulb Ubbelohde instrument using 4.3 meters of 0.5 m m capillary, and shear rates of 25--100 s-1 , and also in a rotating cylinder viscometer similar to the i nst rum ent described by Gill and T h o m p s o n [ 3 3 ] . The r o t o r speed ranged f r om 28 to 300 s/rev, and the shear rates f r o m 0.5 to 0.05 s-I . At the conclusion of each m easurem ent the solution f r o m the cell was assayed for DNA by the m e t h o d of Burton [ 2 3 ] , the recovery always being close to 100%. Circular dichroism measurements were made with a Cary Model 60 recording s p e c t r o p h o t o m e t e r equipped with a Model 6001 CD at t achm ent . Spectra were recorded at 27°C over the wavelength range of 320--220 nm. Experiments were done in 1-cm path length cells with a solution path absorbance at 260 nm o f less than 0.6. The mean residue ellipticity, [ 0 ] , expressed in degree • cm -2 • dmole -~, was based on the DNA nucleotide residue concentration, d eter min ed by ultraviolet absorption at 260 nm, using ext i nct i on coefficients, % , o f 6 8 00 M-~ • cm -1 for DNA and 7950 M -~ • cm -~ for the nucleohistone. The r en atu r a t i on kinetics were measured using the h y d r o x y a p a t i t e technique outlined by Britten and K ohne [ 3 4 ] . Results

The nuclei lyse immediately on mixing with 0.04 M Tris, 0.08 M K2 SO4, pH 7.4, buffer, forming a gelatinous aggregate. In absence of Mg ~÷ or at zero

100

Fraction 3 3 -

Meniscus

Fraction 29 r ReactionM xture Supernatant

/

15

\'~

//

7/

Sedimenlottion

g

e4 I0-

I \ \

(Exp? A) \\ \

05-

\

\

f ~ Experiment B / [

/

\\

:xperirnenlA \\

20

30

40 50 60 Fraction Number

70

80

90

Fig. 1. C h r o m a t o g r a m s of t w o n u c l e o h i s t o n e p r e p a r a t i o n s a n d t r a c i n g s of t h e s e d i m e n t a t i o n v e l o c i t y p r o f i l e s of t h e r e a c t i o n m i x t u r e s u p e r n a t a n t a n d t h r e e c h r o m a t o g r a p h y f r a c t i o n s f r o m o n e of t h e s e . E x p t A (solid line) s h o w s a p r e p a r a t i o n in w h i c h 22% o f t h e n u c l e a r D N A w a s s o l u b i l i z e d while E x p t B ( d a s h e d line) s h o w s a p r e p a r a t i o n in w h i c h 47% was solubilized. T h e b a r s o n t h e c h r o m a t o g r a m for E x p t A i n d i c a t e f r a c t i o n s t h a t w e r e p o o l e d for v i s c o m e t r y w i t h s e d i m e n t a t i o n r u n s m a d e on t h e c e n t r a l f r a c t i o n o f e a c h p o o l (see T a b l e II). T h e s e d i m e n t a t i o n t r a c i n g s r e p r e s e n t a p p r o x i m a t e l y e q u a l c o n c e n t r a t i o n s a n d t i m e s of c e n t r i f u g a t i o n . S e d i m e n t a t i o n c o n s t a n t s ( s 2 0 , w ) of p o i n t s at 20, 50 a n d 80% o f t h e h e i g h t o f e a c h b o u n d a r y w e r e : W h o l e s u p e r n a t a n t : 1 0 1 , 197, 2 7 4 ; F r a c t i o n 33: 111, 1 3 3 , 1 6 5 ; F r a c t i o n 29: 1 4 1 , 176, 2 3 0 ; F r a c t i o n 25: 1 6 2 , 2 3 7 , 324.

time of incubation, no DNA is found in the supernatant. Solubilization kinetics are somewhat variable, but at long times of incubation all of the DNA is solubilized. For this study the incubations were stopped after 30 min to examine a higher molecular weight product. The solubilization varied from 20 to 50%. It is markedly inhibited by Cl-, hence the sulfate buffer used. The product nucleohistone however is freely soluble in buffers containing C1-. Chromatograms always consisted of a leading major and a following minor peak (Fig. 1). Recovery of total ultraviolet absorbing material was close to 100%. The elution volume of the major peak onset was consistently the void volume. Preparations with low percent solubilization gave narrow major peaks (Expt A), those with higher solubilization gave broader major peaks (Expt B). Analysis of fractions of the major peaks showed these to contain approximately, by mass, 1 part DNA, 1.1 part histone, 0.2 part non-histone protein. RNA c o n t e n t is very low or zero. The composition changes abruptly after the main peak. Fractions between the peaks are markedly enriched in protein and RNA, and in the minor peak RNA is the predominant nucleic acid and protein is very high. This material is slow sedimenting, contains the bulk of non-chromatin soluble nuclear contents, and was not examined further.

101

TABLE I SEDIMENTATION

CONSTANTS OF ONE FRACTION IN VARIOUS MEDIA

O n e p e a k f r a c t i o n e l u t e d in S S M V w a s d i l u t e d 5 - f o l d w i t h t h e b u f f e r s listed. S 2 %w at 20, 50 a n d 80% of h e i g h t o f b o u n d a r y is a m e a s u r e o f h e t e r o g e n e i t y In s. Buffer

Composition

Ionic strength (M)

Data listed are extremes of

s20 ,w 20%

50%

80%

179 200 189 200 213 218 222 241 205 218 159 164

222 233 230 236 257 261 269 274 267 268 187 193

277 298 278 290 319 328 316 329 300 327 233 239

SSMV

See f o o t n o t e

0.17

5 runs

A

SSMV without sucrose

0.17

2 runs

B

A w i t h SO42- r e p l a c e d b y C1-

0.17

2 runs

C

B with K + replaced by Na +

0.17

3 runs

D

C w i t h o u t MgC12

0.16

2 runs

C/3

C d i l u t e d 1 --~ 3

0.06

2 runs

S S M V is 0 . 1 2 M s u c r o s e , 0 . 0 3 7 M K 2 S O 4 , 0 . 0 1 9 M Tris, 0 . 0 0 1 M M g S O 4 , 0 , 0 0 5 M E D T A , p H 7.4~ i o n i c s t r e n g t h 0 . 1 7 M, t h e r e a c t i o n m e d i u m a f t e r a d d i t i o n o f E D T A .

The sedimentation patterns of the reaction mixture supernatants show a wide distribution of material f r om near 0 to over 400 S. Fig. 1 illustrates the s distribution of a supernatant of low per c e n t solubilization. No c o n c e n t r a t i o n d e p e n d en ce o f s was f o u n d in the range 15--45 pg DNA/ml and undiluted supernatants, observed by Schlieren optics at DNA concent rat i ons up to 1 mg/ml, gave comparable rates and distribution [ 19]. These supernatants contain all the soluble material of the nuclei. The s distribution of fractions f r om c h r o m a t o g r a p h y are markedly narrowed (Fig. 1). The s values are i n d e p e n d e n t of c o n c e n t r a t i o n in the range of 5--60 pg DNA/ml, showing t hat these high sedimentation rates are n o t the result of a reversible aggregation. T hey are minimally affected by the specific ions in the m e d i u m (Table I). s increased about 15% when SO42- was replaced by C1- at c o n s t a n t ionic strength. No ef f ect followed substitution of Na ÷ for K ÷ or omission o f Mg 2÷ (chelated in any case). Dilution of the medium with water by almost 3-fold caused a 28% fall in s. This must reflect an ionic strengthd e p e n d e n t c o n f o r m a t i o n a l change. These data also illustrate the reproducibility o f the measurements. Later eluting fractions have lower s values. High release preparations have m o r e material eluting late, with the s distribution of the whole supernatant shifted to lower values. This shift may arise from the release of progressively smaller particles or f r om degradation of already released material. Fractions stored at 4°C exhibit a slow decline in s, a b o u t 10% in one week. In the case of Fraction 25, E x p t A (Fig. 1), a very early fraction of a low release preparation, the s distribution shows a marked widening at the high s end. This p r o b ab l y reflects the presence of very large particles which are n o t fractionated on Sepharose 2B. S ed imen tation data f r om three preparations are c o m b i n e d with viscosities

102 TABLE

II

PROPERTIES

OF SOLUBLE

NUCLEOHISTONE

Experhnent Percent solubilized

A 22%

Fraction

25

29

33

28

37

22

31

37

237 12.8 5.6 214

179 6.2 2.7 95

133 3.2 1.4 45

252 1.0 0.4 64

142 0.6 0.3 23

250 4.5 2.0 138

120 2.8 1.2 36

70 1.4 0.6 11

52

47

30 21 95

25 13 70

No.

Nucleohistone o S20,w Iv/], dl/g DNA [~], dug NH* Mol. wt X 10 -6**

Nucleohistone in 2 M N a C l [77], d l / g D N A 206

B 47%

C 40%

DNA

o S20,w Mol. wt X 10 -6 [~], dug***

32 22 103

32 24 105

23 11 61

18 6 39

Ratios Mol. w t N H Mol. w t D N A

9.7

4.5

3.5

5.8

3.3

1.8

[q] DNA [~] NHt

8.0

15

22

23

22

28

[7)] N H ( 2 M N a C l ) t [q]NHt

16

8.4

15

* Calculated on the basis of DNA/protein = 1/1.3. ** From the Scheraga--Mandelkern [ 3 5 ] e q u a t i o n (/3 = 2 . 1 1 . : 1 0 6 , v = 0 . 6 6 5 ) . S i n c e t h e p a r t i c l e s are e l o n g a t e d o r i r r e g u l a r t h e s e m o l e c u l a r w e i g h t s are u p p e r l i m i t s . T h e c a l c u l a t i o n is s o i n s e n s i t i v e t o s h a p e h o w e v e r t h a t it is u n l i k e l y m o l e c u l a r w e i g h t h a s b e e n o v e r e s t i m a t e d b y m o r e t h a n 2 0 % . It is also insensitive to other choices for ~ and for nucleohistone/DNA mass ratio, within plausible limits. *** Calculated from molecular weight from s. t These viscosities of nucleohistone are e x p r e s s e d in d l / g D N A .

and interpreted in terms of molecular weights in Table II. The specific viscosity o f n u c l e o h i s t o n e fractions measured in the capillary viscometer at shear rates from 25 to 1 0 0 s -1 s h o w e d a marked d e p e n d e n c e on shear rate. The reduced viscosities, extrapolated to zero shear, for three fractions of one e x p e r i m e n t are included in Table II ( E x p t C). In the rotating cylinder viscometer, the reduced viscosities were i n d e p e n d e n t of b o t h shear and c o n c e n t r a t i o n . The intrinsic viscosities are expressed in Table II in t w o forms, the first based o n the D N A c o n c e n t r a t i o n , the s e c o n d on the n u c l e o h i s t o n e c o n c e n t r a t i o n assumed to be 2.3 times the D N A . Inspection o f these results s h o w s that the intrinsic viscosities d e c l i n e as m u c h as 3-fold from early to late eluting fractions o f the main peak. Further, even at fixed elution v o l u m e there is a large variation, in intrinsic viscosity, b e t w e e n preparations. N o change in intrinsic viscosities was f o u n d after 3 days of storage at 4 ° C. The viscosities are strikingly l o w , especially w h e n c o m p a r e d to those o f the R N A isolated from the same n u c l e o h i s t o n e fractions (Table II). It is n o t necessary to isolate the D N A to d e m o n s t r a t e that point. N u c l e o h i s t o n e fractions of E x p t A were diluted

103 F2b F2o2

SALT WASHED NUCLEI

F2ol

REACTION MIXqURE

REACTION MIXTURE

SE

A

SE

FRACTIONS

Fig. 2. Tracings o f e l e c t r o p h o r e s i s gels. Histories w e r e e x t r a c t e d into 0~2 M H 2 S O 4 f r o m rabbit t h y m u s n u c l e i w a s h e d w i t h 0 . 1 5 M NaCl, 0 . 0 1 5 M citrate, 0 . 0 0 5 M N a H S O 3 , p H 7.0, f r o m a r e a c t i o n m i x t u r e s u p c r n a t a n t , and f r o m p e l l e t e d n u c l e o h i s t o n e f r a c t i o n s . T h e y w e r e p r e c i p i t a t e d w i t h cold e t h a n o l , rediss o l v e d in 0.9 M a c e t i c acid and s u b j e c t e d to gel e l e c t r o p h o r e s i s a c c o r d i n g to P a n y i m and C h a l k l e y [ 2 8 ] . U p p e r : H i s t o n e s e x t r a c t e d f r o m u n d e g r a d e d c h r o m a t i n , labeled w i t h the n o m e n c l a t u r e o f Phillips and J o h n s [ 3 6 ] as i d e n t i f i e d b y P a n y i m and C h a l k l e y . S e c o n d : H i s t o n c s e x t r a c t e d f r o m a r e a c t i o n m i x t u r e s u p e r n a t a n t w h i c h c o n t a i n e d 0 . 0 0 5 M N a H S O 3 during the i n c u b a t i o n ( p H = 7.4). Third: H i s t o n e s extracted from a reaction mixture supernatant (without NaHSO3). Bottom: Histones from nucleohistone fractions.

5--50-fold with 0.1 M KC1, 0.02 M Tris--HC1, 0 . 0 0 5 M citrate pH 7.4 with sufficient NaC1 for a final concentration of 2 M. The reduced viscosities are s h o w n in Table II. They are 8 - - 1 6 times those in 0.1 M KCL, 0.02 M Tris--HC1, 0.005 M citrate pH 7.4. Increases up to 50-fold have been seen in some preparations. Sedimentation constants of the D N A isolated from each fraction are given in Table I! with the calculated molecular weights and intrinsic viscosities. The molecular weight of the D N A declines from early to late eluting fractions. Early fractions contain four D N A molecules in each nucleohistone particle, while later fractions must typically have only one. Sedimentation boundaries of D N A even at low concentration were always less broad than those of the nucleohistone, another indication of the c o m p l e x i t y of nucleohistone structure. Histories from nucleohistone fractions were examined by gel electrophoresis in urea--acetic acid, and compared with histones of the incubation mixture supernatants and o f saline-washed nuclei. Typical tracings are shown in Fig. 2. No difference was found between histones of early and late eluting

104 I0-

5~

~

~

i

i

~

DNA

0 - - E -5-

g

-fO-

E -15-

x

-20-

-25-

-30-

-35 -

, 220

i

, 240

,

i

,

260

,

F

280

, 300

,

, 320

">,.(nm)

Fig. 3. Circular

dichroism

spectra

of three

undiluted

nucleohistone

middle, and late areas of the major peak and of rabbit thymus run in 0.1 M KC1, 0.02 M Tris-HCl, 0.005 M citrate pH 74.

DNA

chromatography purified

fractions

by pelleting

from

early,

in 4 M CsCI, all

f r a c t i o n s . Comparing nucleohistone from c h r o m a t o g r a p h y with the undegraded ch r o matin, we not e the appearance o f two new bands, one between F1 and F3, and a m uc h smaller one leading F2al. These bands are probably p ro du cts of proteolysis of histones; similar bands have been r e p o r t e d by Panyim and Chalkley [ 2 8 ] . Following those authors we added 0.005 M NaHSO3 to our incubation mixture in an a t t e m p t to inhibit proteolysis. The e x t e n t of solubitization was the same as in control incubations. Krueger and Allison [21] also f o u n d no inhibition of release. The form at i on of the band leading F2al was inhibited, but the band between F1 and F3 was not. Fig. 3 shows the CD spectra o f three nucleohistone fractions and of rabbit t h y m u s DNA. The difference between fractions is n o t significant. The maxim u m ellipticity of DNA was f o u n d to be 9000 degree • cm -2 • dmole -1, of the nucleohistone fractions, 2250. Our experience indicates t ha t the nucleohistone has no t e n d e n c y to aggregate if gently handled. However, certain t reat m ent s abruptly precipitate the nucleohistone or a large fraction of it, particularly mild hom ogeni zat i on and dissociation--reassociation by adding salt, then diluting with water. Fractions f r o m c h r o m a t o g r a p h y were hom oge ni z e d ice-cold in a Potter--Elvejem h o m o g e n i z e r at low rev./min. H om oge ni z a t i o n induced marked turdibity. After centrifugation at 2300 rev./min for 20 min only 18-20% of the original A2 a 0 n m was in the supernatant. Such centrifugation pelleted no measurable a m o u n t f r o m controls. H o m o g e n i z a t i o n of the pellets in their supernatants, even at high speeds, gave no redissolution. NaC1 was added to reaction mixture

105

x "%%•

30 -L

~

\

\\

~,

o

m

•

o

40-

--_

w

~o70-

80-

90

,02

,i,

,

,~ Cot

,~2

,0+

,14

(M secll)

Fig. 4. Kinetics

of renaturation of DNAs measured by the hydroxyapatite technique of Britten and Kohne DNAs were sheared to 250 000 mol. wt, heat denatured, chilled, diluted with 0.12 M phosphate, pH 6.8 to convenient concentrations a n d i n c u b a t e d a t 6 0 ° C f o r t i m e s o f 8 rain t o 4 . 5 d a y s . D N A e l u t i n g from hydroxyapatite at 6 0 ° C w a s t a k e n t o b e t h e d e n a t u r e d f r a c t i o n , t h a t r e m a i n i n g a n d e l u t e d at 9 5 ° C the native fraction. :--~;, DNA from whole rabbit thymus chromatin; o--e, DNA from solubilized nucleohistone A DNA from unsolubilized pellet from the same incubations. The upper ~olid line represents t h e s e d a t a . ~ a n d l o w e r s o l i d l i n e are o u r r e s u l t s o n c a l f t h y m u s D N A . - - -, c a l f t h y m u s D N A , r e s u l t s o f B r i t t e n a n d K o h n e [ 3 4 ] . T h e p e r c e n t n a t i v e D N A in t h e h o r i z o n t a l p o r t i o n o f t h e c u r v e s is t a k e n t o b e the percent repetitive sequences. [34].

supernatants to i or 2 M. The solutions remained clear but their viscosity rose abruptly, forming an elastic gel. Water was added to 0.15 M ionic strength. Conspicuously stringy precipitates formed immediately. After centrifugation the supernatants contained 16--30% of the original A260 Controls diluted with 0.15 M NaC1 to the same volumes became slightly turbid and their supernatants contained 64--102% of the original ultraviolet absorption. Nucleohistone similarly prepared and purified on a Sephadex G-200 column with minimal dilution (DNA concentration approx. 1 mg/ml), kindly provided by Dr R.C. Krueger, gave a bulky, stringy precipitate when treated similarly. In order to determine if the autolysis preferentially solubilized repetitive or unique DNA sequences, renaturation rates were measured on DNA isolated from reaction supernatants and pellets, from whole rabbit thymus chromatin, and as further control, calf thymus DNA. Results are shown in Fig. 4. Our measurements of calf thymus DNA show 40% repetitive sequences, in agreement with the results of Britten and Kohne [ 3 4 ] . The slight shift in the direction of more rapid renaturation may arise from a slightly smaller molecular weight to which our DNA was sheared. In rabbit thymus DNA from whole chromatin we find 28% repetitive sequences. DNA from the reaction mixtures pellets or supernatants did not significantly deviate from this value. n m

•

Discussion The most distinctive characteristics are its solubility in 0.15 M buffers, its compact configuration, its high molecular weight, and its CD spectrum. Its

106 gross co mp o s ition and thermal denaturation curves (19 and data n o t shown) are similar to those of other preparations. The molecular weight of its DNA is higher than most reports of DNA isolated from shear-solubilized chromatin. The renaturation rate data indicate the DNA is n o t distinctive in its base sequences. The histone distribution is near normal but shows evidence of some proteolysis. This mixture of normal and distinctive properties must result from the unique man n er of its preparation, a procedure which utilizes tissue degradative enzymes but avoids shearing and pelleting. The essential reactions of the solubilization process have n o t been defined. Cleavage of the DNA must be necessary and does occur. A proteolysis or a protein removal process may also be necessary. The appearance of two new bands is suggestive of the specific cleavage of one or two histones, but we c a n n o t state which are being cleaved. The five principal histone bands are present in near normal amounts. The DNA solubilized in 30 min varied from about 20 to 50%. This variability may arise f r om the condition of the gel in which the process occurs. On mixing the nuclei with buffer, the nuclei lyse and their contents aggregate, forming a loose gel which contracts s om ew hat on swirling and more so on vigorous mixing. Greater solubilization occurs when the gel is more extended. In the denser gels nucleohistone particles, cleaved free by the nucleolytic and perhaps p r o t e o l y t i c reactions, may be ent r a pp ed and only slowly find their way into the supernatant. Vendrely [37] observed the autolysis (w i t hout Mg 2÷) of chromatin exposed to shear in several prior washings, and found very much slower solubilization and an extensively depol ym eri zed product. The h y d r o d y n a m i c data on nucleohistone given here include sedimentation constants and molecular weights higher, and intrinsic viscosities lower, than any we have seen in the literature. The ratio of the viscosity of the nucleohistone to that of the DNA isolated from it is also a new extreme. The very low e!lipticity at 275 nm, only one quarter t hat of DNA, shows that the stacking g eo metr y of the DNA bases has been disturbed, consistent with a c o n t o r t e d configuration of the DNA strands. This is a p r o p e r t y characteristic of chromatin but displayed here in greater degree. These indications of a very c o m p a c t configuration are consistent with the electron micrographs of Krueger and Allison [ 2 1 ] . Whether or n o t this state is indeed a feature retained from the native state of chromatin, it is a remarkable example of a c o n t o r t e d configuration imposed on DNA by associated proteins. The h e t e r o g e n e i t y of size of the nucleohistone is to be expect ed in the p r o d u c t o f a degradative process especially in view of the necessarily complex structure of chromatin itself. The variability of the intrinsic viscosities deserves more c o m m e n t . T ha t [r~] is lower in the later eluting, lower molecular weight and lower s, fractions is consistent with the electron micrographs of Krueger and Allison [21] and indeed with m a ny elongated, coiled, or folded structures. That [7] varies between preparations, when particles of similar molecular weight are compared (by interpolation between fractions in Table III), is surprising. It demands separate processes controlling size and configuration. Among the factors to be considered are DNA cleavage and the various modifications that may occur to the histones (proteolysis, p h o s p h o r y l a t i o n or

107 dephosphorylation, acetylation, etc.), also the distribution of enzymes and perhaps of histones between the gel and the supernatant. That nucleohistone has so often been reported insoluble in 0.15 M salt, and is almost universally studied in low salt media, is an obstacle to its functions. How can DNA synthesis, for example, proceed in the solid state? The present material is freely soluble until sheared, and then is precipitated and resists dispersion in the original medium. This precipitation shows that its solubility must depend on rather fragile details of structure, and is not wholly the result of deproteinization or other degradation. Chromatin itself must consist of very large and fragile molecules. Any process solubilizing chromatin must necessarily be degradative. The possibility that conventional methods degrading it by shear alter its structure in subtle ways must be considered. The present material has properties which are unusual in the direction we would expect of chromatin in the cell nucleus. Experiments are in progress to explore the structural origin of these properties. Alternate methods of solubilizing chromatin, which avoid shear or pelleting, particularly one using defined enzymes, would be enlightening, if such a system could be found.

Acknowledgements We are indebted to Dr David J. Cox for the use of the circular dichroism instrument and his assistance in measuring the spectra. This work was supported by The Robert A. Welch Foundation, grant n u m b e r AQ-465.

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