volume 13 Number 10 1985 Nucleic Acids Research · Nucleic Acids Research cation, in the presence of histone variants and nonhistone proteins (for re-viex see 1, 2, 11) and in soae

volume 13 Number 10 1985 Nucleic Acids Research

Primary organization of nudeosomai core particles is invariable in repressed and active nuddfrom animal, plant and yeast cdls

Sergei G.Bavykin, Sergei I.Usachenko1, Alia I.Lishanskaya*, Valentin V.Shick, AlexanderV.Belyavsky, Igor M.Undritsov, Alexei A.Strokov, Irina A.Zalenskaya1 and Andrei D.Mirzabekov*

Institute of Molecular Biology, USSR Academy of Sciences, Moscow 117984, 'Institute of BioorganicChemistry, Academy of Sciences of the Uzbek SSR, Tashkent 700000, 'Institute of Nuclear Physics,USSR Academy of Sciences, Leningrad 188350, and 'Institute of Cytology, USSR Academy ofSciences, Leningrad 194064, USSR

Received 12 March 1985; Accepted 20 April 1985

ABSTRACT

A refined aap for the linear arrangement of histones along DMA in nucleo-soaal core particles hai been determined by DNA-protein crosslinking. On onestrand of 145-bp core DNA, histones ire aligned in the following orderi(5') H2B a o,3o-H4Bo,*B-H37o,.B,»o/H4,,-H2B,oo.iio-H2Ai,,-H3i3B. >«o/H2A 1,o

(3'>(the subscripts give approxiaate distance in nucleotides of the aain histonecontacts fro* the 5'-end). Hence, the histone tetraaer (H3,H4), and twodi«er« (H2A-H2B) are arranged on double-stranded core DNA in a syaaetricaland rather autonomous nay: H2A/H3-(H2A-H2B)-(H3,H4) a-(H2B-H2A)-H3/H2A.The priaary organization Has found to bs very sieilar in cor* particlesisolated froa repressed nuclei of sea urchin spern and chicken erythrocytes,froo active in replication and transcription nuclei of Drosophila eabryosand yeast and froa soaatic cells of lily. These data ihoN that (i) the corestructure is highly conserved in evolution and d i ) the overall inactivati-on of chroaatin does not affect the arrangeaent of histones along DNA andthus does not seea to be regulated on this level of the core structure.

INTRODUCTION

Nucleosoaal core particle is the basic unit of chroaatin structure. It

consists of about 14S base pairs (bp) of DNA and a histone octaaer contain-

ing tno aolecules tach of histones H2A, H2B, H3 and H4. Together with his-

tone HI and spacer DNA of variable length the core particles fora nucleoso-

aes (1, 2 ) . The X-ray and electron aicroscopy studies of core crystals and

ordered aggregates of histone octaeers performed by Klug's group have alre-

ady attained 7 fi resolution and should hopefully reach 5 8 (3-6). In our

laboratory we used specific DNA-protein crosslinking combined Hith tno-di-

aensional gel electrophoresis of the crosslinked material to deteraine the

sequential arrangement of histones along DNA (priaary organization) in core

particles and Hl-containing nucleosomes (7-9). Recent laproveaents in this

technique and the use of cores from different sources have enabled us in

this paper to refine our previous map for the primary organization of nuc-

leosoaal core particles (7, 10).

Nucleosoaal cores, although invariable in all eukaryote6, represent a

highly heterogeneous population of particles that differ in histone aodifi-

© IR L Press Limited, Oxford, England. 3439

Nucleic Acids Research

cation, in the presence of histone variants and nonhistone proteins (for re-

viex see 1, 2, 11) and in soae physical properties (12-14). It appears that

this heterogeneity say be both the cause and a result of aany functional

states of chroaatin.

The corei froa transcribed chroaatin regions auit have soae peculiariti-

es in structure since they are preferentially digested by DNAase I (15, 16),

can be enriched in HH6 14 and 17 proteins (17) and ubiquitin conjugated to

histone H2A (IB), show an increased affinity for HH6 14 and 17 proteins

(19) and for RNA polyaerase II (20) and contain hyperacetylated histone

tpecies (reviewed in 2 1 ) . The cores isolated froa transcribed ribosoaal

chronatin of Phytarue polycephalua show exposed H3 cy6tein residues, and

in electron aicrographs they look like partly unfolded bipartite particles

(22) .

Replication and transcription of DNA in chroaatin should apparently af-

fect the D N A - h n t o n e contacts. To cite a few extreae exaaples, it Has shown

that histories got reaoved froe actively transcribed ribosoaal genes (23),

heat shock genes (24) and froo the 5 '-regulatory, DNAase I hypersensitive

gene regions (24, 2 5 ) . It i> clear thtrefore that a c o a p a n s o n of the ar-

rangeacnt of DNA-histone contacts in nucleosoaes of functionally different

chroaatin regions could be of essential interest. The "protein iaage* hybri-

dization technique developed in this laboratory (24) enables one to coapart

the priaary organization of nuclaosoaes in various genes and within differ-

ent regions of the itte gent, and such experiaents are now in progress.

In this paper, we have cade use of another approach and coapared the core

priaary organization in repressed nuclei of sea urchin spera and chicken

erythrocytes and in highly active in replication and transcription nuclei of

Drosophila eabryos, lily buds and yeast. Our results show that the priaa-

ry organization of core particles is nearly identical in all three higher

eukaryott kingdoas. The core organization is also very siailar in both ac-

tive and repressed nuclii which suggests that the overall repression of

chronatin is unlikely to be regulated on the level of the arrangaaent of

histones along core DNA.

MATERIALS flND METHODS

Isolation of nuclei

Nuclei froa sea urchin spers (Strongi1ocentrotus interaedius) were pre-

pared as in (26).

Yeast nuclei were isolated according to the aethod (27) with soae aodifi-

cations. In particular, spheroplasts were aade froa exponentially growing

344O


culture of Saccharoayces cerevisiae strain DC5 using snail gut enzyaes

instead of Zysolyase. After centrifugation in Percoll gradient, the nuclei

tiere washed three tises in 0.5X NP-40, 50 all Na-phosphate (6.8) by centri-

fugation at 17,500 g for 15 Bin, three tiaes in the saae buffer containing

2 aM EDTA without detergent and three tiaes in 0.35 M NaCl, 2 iM EDTA,

10 «M Na-phosphate (pH 6 . 8 ) .

Isolation of nuclei and chroaatin froa Drosophila eabryos and chicken

erythrocytes was perforaed as described in (7) and (28).

Nuclei of lily (Li 1iua Candida, L.) were prepared as will be described

elsewhere (Bavykin et al., in preparation). Sepals froa buds (60 g) were

powdered with dry ice. The ice was evaporated at -20°C, the hoaogenate

was stirred in 1.5 1 of buffer 1 containing 21 Triton X-100, 0.3 M sucrose,

0.5 aM diisopropyl f1uorophosphate, IX diaethylsulphoxide, 50 aH Tris-Cl

(pH 7.6) for 12 hr at 4°C, filtered through several layers of cheese-

cloth and centrifuged for 10 ain at 300 g. The pellet was waihed twice in

350 al of the saae buffer containing IX Triton X-100, 0.4 H sucrose, cent-

rifuged for 10 ain at 500 g, suspended in 100 al of buffer 1 containing

0.25X Triton X-100, 0.25 M sucrose, layered over three voluaes of the

sa«e buffer containing 0.25X Triton X-100, 0.5 H sucrose and centrifuged

for 20 ain at 1000 g. The nuclei were washed once with 50 aH Tris-Cl (pH

7. 6 ) , centrifuged for 10 Bin at 500 g and finally waihed three tiaes in

0.35 H NaCl, 2 aM EDTA, 10 aH Na-phosphate (pH 6.8) by centrifugation at

5000 g for 10 Bin.

Histone-DNA crossl inki nq in sea urchin spera, yeast and lily nuclei

DNA-histone crosslinking was conducted as described in (7,29). Nuclei

(2000 Aito units) were suspended in 50 al of 0.5 aH EDTA, 0.5 aH diiso-

propyl f luorophosphate, IX diaethylsulphoxide, 50 aH Na-cacodylate (pH 7.0)

and diaethylsulfate was added to a final concentration of 4.3 aH under vigo-

rous stirring. The DNA was methylated at 4°C for IB hr. The aethylated

nuclei were waihed twice with 80 eH NaCl, 5 aH EDTA, 20 aH Na-phosphate (pH

6.8). D e p u n n a t i o n of aethylated bases in DNA was perforaed by incubation

of nuclei at 45°C for 8 hr in 50 al of the above buffer containing

0.5 aH diisopropyl fluorophosphate added in diaethylsulphoxide. The nuclei

wire washed twice in 100 aH Na-phosphate (pH 6.8) and suspended in the saae

solution where the h i s t o m - D N A aldiaine bonds forced were reduced by adding

1/10 voluoe of freshly prepared 250 »H Na-borohydride and incubating at

4°C for 30 ein in the dark. Finally, the nuclii were washed successively

with 100 aH, 50 aH and 20 aH Na-phosphatt (pH 6.8).

3441


Isolation of core particles froa crosslinked nuclei of sea urchin spera,

lily and yeast

Crosslinked nuclei from sea urchin spera or lily buds Here suspsnded,

respectively, in 0.4 and 1.5 aH Ca C l a , 10 oM Tris-Cl (pH 8.0) at a con-

centration of ISO A n o units/al and digested Hith 85 units/al aicrococcal

nuclease (Worthington) for 20 ain at 37°C. The reaction Has terminated by

adding EDTfl to 5 B M . Highly purified core particles Mere isolated froa the

digest by electrophoresis in a polyacrylanide gel (7, 3 0 ) . Crosslinked

yeast nuclei (20 A 3 4 O units/il) Here incubated with aicrococcal nuclease

at a concentration of 15 u m t s / i l in 0.5 B H C a d i , 20 an Na-phosphate

(pH 6.8) for 20 ain at 37°C, and the nucleosoae core particles Here puri-

fied by sucrose gradient centrifugation in 1 B H EDTA, 20 aM Na-phosphate

(pH 6.8) for 35 hr at 75,000 g.

Isolation and crosslinking of cores frog chicken erythrocyte and Drosophi-

la eabryo chroaatin

The core particles froe uncrosslinked chroaatin of chicken srythrocytes

and Drosophila eabryos Here prepared according to (7). First, histone HI

Has reaoved froa chroaatin on D O H B X A6-50-X2 ( 3 1 ) , then the chroaatin Has

treated with aicrococcal nuclease and the core particles Here purified by

sucrose gradient centrifugation. Histones Here crosslinked to DNA in isola-

ted core particles as recoaaended in (7).

Enrichaent and labeling of crosslinked DNft-histone coaplexes

Host free histones were reaoved froa the crosslinktd a t t e n a l before its

labeling nith l " I or " P by precipitating DNA together Hith crostlinked

histones as cetyl triaethylaaaoniua (Cetavlon) salti ( 7 ) .

Thin the crosslinked coaplexes Here labeled at tyrosine residues of his-

tones Hith 1 1 B I as described (32), taking 0.5 aCi of N a ' " I for libeling

1 A a « o units of the coaplex (the aaount used for one two-diaensional elec-

trophoresi s) . DNA in the crosslinked coaplex Has labeled at the 5' end with

" P according to ( 3 3 ) . The coaplex (1 A z * o units) in 0.03 al of 10 aH

HgCl,, 10 ad dithiothreitol, 0.1 aH speraidine, 40 aH BICIN (pH 9.0) Has

incubated at 37°C for 1.5 hr Hith 2 units of polynucleotids kinase and

0.5 aCi of (-y-"P>ATP. This aoount Has sufficient for 1 to 4 tno-

diaensional electrophoreses, depending on the gel size. Next, aost of the

free DNA Has reaoved froa the crosslinked coaplex by hydroxylapatite chroe-

atography of SDS-protein coaplexes (34) as described in (24) Hith soae aodi-

fications. 3 a P - l a b e l e d crosslinked aaterial (1 flj.o units) Has dissolved

in 0.03 al of 17 SDS, 10 aH dithiothreitol, 5 aH Na-phosphate(pH 6.4) and in-

cubated at 100°C for 1 ain to denature DNA. Then 0.27 al of 0.1X SDS, 5 aH

3442


N a - p h o s p h a t e was a d d e d , and the s o l u t i o n w a s loaded o n t o a t h e r o o s t a t e d at

30 ° C c o l u a n c o n t a i n i n g 0.5 al o-f h y d r o x y l a p a t i t e s y n t h e s n e d in s i l i c a g e l

( 3 5 ) ; 3 0 a m l a t e r , the c o l u a n H a s w a s h e d with 5 al of 0.1Z S D S , 5 aM N a -

p h o s p h a t e (pH 6 . 4 ) ; the f r e e DNA was r e a o v e d by 0.11 S D S , 0.15 H N a - p h o s -

p h a t e (pH 6 . 4 ) , and the c r o s s l i n k e d D N A - p r o t e i n c o a p l e x was e l u t e d with 0.1X

S D S , 0.5 M N a - p h o s p h a t e . The e l u t i o n w a s a o n i t o r e d by r a d i o a c t i v i t y a e a s u r e -

a e n t s . The a a t e n a l was d i a l y s e d at 4°C -for 12 hr a g a i n s t 0. 1Z S D S ( c h a n -

ged t w i c e ) , DNA f r a g a e n t s f r o o D N A a s e I d i g e s t of rat liver n u c l e i (36) w e r e

a d d e d to the 0.1 ag/al c o n c e n t r a t i o n , and the c r o s s l i n k e d DNA was p r e c i p i t a -

ted with 3 v o l u e e s of et h a n o l f r o a 0.2 M N a C l .

T w o - d i a e n s i onal e l e c t r o p h o r e s i s of c r o s s l i n k e d D N A - h i s t o n e c o a p l e x e s

All e l e c t r o p h o r e t i c s e p a r a t i o n s w e r e p e r f o r a e d as d e s c r i b e d e a r l i e r (7)

wit h t h e a o d i f i c a t i o n s s p e c i f i e d b e l o w . T h e L a e a a l i b u f f e r s y s t e a (37) w i s

us e d , 7 M ur e a was p r e s e n t when i n d i c a t e d .

E l e c t r o p h o r e s i s in the f i r s t d i r e c t i o n w a s c o n d u c t e d in t h i n n e r g e l s

(0.6 a a ) w h i c h e n a b l e d us to s h o r t e n s i g n i f i c a n t l y the f u r t h e r t r e a t a e n t of

gel s t r i p s and thus l a p r o v e r e s o l u t i o n in the s e c o n d d i r e c t i o n . A gel s t r i p

c o n t a i n i n g ' " I - h i s t o n e s w a s w a s h e d s u c c e s s i v e l y in 661 f o r a i c a c i d , 21 d i -

p h e n y l a a i n e in 661 f o r a i c a c i d , then i n c u b a t e d in the latter s o l u t i o n for 15

•in at 7 0 ° C , wa s h e d four t i a e s in 66X f o r o i c a c i d , s e v e r a l t i a e s in w a t e r

until n e u t r a l pH, and f i n a l l y two t i a e s in 0.IX S D S , 0.125 M T r i s - H C l (pH

6 . 8 ) . All w a s h e s w e r e d o n e on a a a g n e t i c s t i r r e r for 10 ain e a c h .

H i s t o n e s in a f i r s t - d i a e n s i o n gel s t r i p c o n t a i n i n g " P - l a b e l e d c r o s s -

l i n k e d a a t e r i a l w e r e h y d r o l y s e d in t h e gel in the s e c o n d d i r e c t i o n w i t h P r o -

n a s e (3B) as d e s c r i b e d (7) e x c e p t that t h e r e w a s no need to s w i t c h off the

cu r r e n t d u r i n g h y d r o l y s i s .

6el d i a e n s i o n s and oth e r e l e c t r o p h o r e t i c p a r a m e t e r s are g i v e n in the

f i g u r e l e g e n d * .

R E S U L T S

C h r o a a t l n

C h r o a a t i n of sea u r c h i n s p e r a and c h i c k e n e r y t h r o c y t e s is a l a o s t c o a p l e -

tely r e p r e s s e d and p a r t i c i p a t e s n e i t h e r in r e p l i c a t i o n nor in t r a n s -

c r i p t i o n . On the o t h e r h * n d , c h r o a a t i n f r o a D r o s o p h i l a e a b r y o s , lily b u d s

and e s p e c i a l l y f r o a yeast is e f f e c t i v e l y i n v o l v e d in t h e s e p r o c e s s e s , with

all n u c l e o s o o e s u n d e r g o i n g r e p l i c a t i o n in ea c h c y c l e of cell d i v i s i o n and

at least p a r t l y p a r t i c i p a t i n g in t r a n s c r i p t i o n . H o w e v e r , it is d i f f i c u l t to

e s t i a a t e q u a n t i t a t i v e l y what p o r t i o n of n u c l e o s o a e s is i n v o l v e d in t r a n s -

c r i p t i o n . In the e x p o n e n t i a l l y g r o w i n g y e a s t used in th i s s t u d y , DNA is

t r a n s c r i b e d by it least 4 0 Z ( 3 9 ) , and all c h r o o a t i n s h o w s the " a c t i v e " p r o -

3 4 4 3


H7BL

™~ <—> HISN4M« « • ' ^ H4 H4

F i g u r e 1. E l e c t r o p h o r e s i 3 of histories f r o a s e a u r c h i n s p e r a ( a ) , c h i c k e ne r y t h r o c y t e ( b ) , y e a s t (c) and lily b u d (d and e - s h o r t and long e x p o s u r e )c o r e p a r t i c l e s in 151 pol y a c r y l a s i d e gel in t h e p r e s e n c e of 0. II S D S (37)

p e r t i e s u p o n d i g e s t i o n w i t h D N A a s e I and - t r a c t i o n a t i o n ( 4 0 ) ; H e c o n c l u d e

t h e r e f o r e t h a t at l e a s t a c o n s i d e r a b l e p a r t of t h e y e a s t c o r e s o r i g i n a t e

f r o o t r a n s c r i p t l o n a l 1 y a c t i v e c h r o a a t i n .

A n o t h e r a m of t h i s s t u d y H a s to c o s p a r e t h e p r i a a r y o r g a n i z a t i o n of

c o r e n u c l e o s o a e s i s o l a t e d froa d i f f e r e n t s o u r c e s s p a n n i n g all t h r e e h i g h -

er e u k a r y o t e k i n g d o a s . S e a u r c h i n s p e r a and li l y b u d s s e e a e d p a r t i c u -

l a r l y s u i t e d for t h e p u r p o s e of e s t a b l i s h i n g a p o s s i b l e c o r r e l a t i o n b e t w e e n

t h e p n o a r y o r g a n i z a t i o n of n u c l e o s o a e s and c o r e h i s t o n e s s t r u c t u r e . T h e

s t r i k i n g f e a t u r e of sea u r c h i n s p e r a a l c h r o a a t i n is t h e p r e s e n c e of s e v e r a l

v a r i a n t s of h i s t o n e H 2 B which a o l e c u l i s c o n t a i n a c o n s i d e r a b l y e x t e n d e d N -

t e r a i n a l p a r t w i t h an a d d i t i o n a l c l u s t e r of 4-5 l y s i n e and s e v e r a l a r g i n i n a

r e s i d u e s ( 4 1 ) . T h e s e s p e r s a l H 2 B h i 3 t o n e s h a v e a d e c r e a s e d n o b i l i t y and a r e

b e t t e r r e s o l v e d f r o m o t h e r h i s t o n e s by e l e c t r o p h o r e s i s in S D S g e l s . W e h a v t

t a k e n a d v a n t a g e of t h i s fact t o try a n d a c h i e v e a h i g h e r r e s o l u t i o n in s a p -

p i n g h i s t o n e c o n t a c t s a l o n g c o r e D N A . In l i l y , as Hel l a s in o t h e r p l a n t * ,

H 2 A a n d H 2 B h i s t o n e s (except o n e H 2 A s u b t r a c t i o n in l i l y ) a r e u s u a l l y l o n g e r

and m i g r a t e s l o w e r in S D S g e l s t h a n t h e s a a e h i s t o n e s f r o a a n i a a l c e l l s

( F i g u r a 1 ) . T h e p o s i t i o n i n g of lily h i t t o n t s H a s d e t e r a i n e d w h e n t h e S O S

e l e c t r o p h o r e t i c s y s t e e H a s u s e d in t h e s e c o n d d i r e c t i o n a f t e r t h e h i i t o n a i h a d

b e e n e l e c t r o p h o r e s e d in a T r i t o n - a c e t i c a c i d - u r e a gel (42) in t h e f i r s t d i -

r e c t i o n (not s h o w n ) .

3 4 4 4


F i g u r e 2 . T w o - d i m e n s i o n a l sequencing gel e l e c t r o p h o r e s i s of s i n g l e - s t r a n d e d ," P - l a b e l e d DNA c r o s s l i n k e d to h i s t o n e s in sea urchin speraal c o r e s .E l e c t r o p h o r e s i s in the first d i r e c t i o n Has carried out in 7 M urea under DNAde n a t u r i n g c o n d i t i o n s in 17X p o l y a c r y l a a i d e slab gel (16 5 x 3 6 5 x 0 . 6 aa) at aconstant current of 3 aA and 7 aA for the c o n c e n t r a t i n g and separ a t i n g g e l ,r e s p e c t i v e l y , for 30 n n until broaphenol blue ran about 1.5 length of thesep a r a t i n g g e l . After digestion of h u t o n e s Mith P r o n a s a in the gel ( 3 8 ) ,e l e c t r o p h o r e s i s was continued in the second d i r e c t i o n in 151 p o l y a c r y l a a i d eslab gel (300x400x1 ••) c o n t a i n i n g 7 M urea at a co n s t a n t current of 10 aAand 20 aA for the c o n c e n t r a t i n g and s e p a r a t i n g g e l , r e s p e c t i v e l y , untilb r o a p h e n o l blue reached the bottoa of the g e l . The broken lines show p o s i -tion in the gel and the figures give the length of e t h i d i u a - b r o a i d e - s t a i n e dDNA f r a g m e n t s froa D N A a s e I digests of rat liver n u c l e i . Their p r e c i s e va-lues are 20, 3 1 , 4 1 , 5 2 , 63, 73, 83, 93, 103, 113, 124, 133 lnd 142 bases(46, 4 7 ) . The p o s i t i o n s of " P - l i b e l e d DNA f r a g a e n t s c r o s s l i n k e d to dif-ferent h u t o n e s and arranged on se p a r a t e d i a g o n a l s Here revealed by au t o r a -d i o g r a p h y and are indicated by solid l i n e s . The ex t r e a e right diagonal c o n -tains u n c r o s s l i n k e d DNA f r a g a e n t s . At the left is the a u t o r a d i o g r a a of thetop part of the gel after a shorter e x p o s u r e .

Except speraal H2B and lily H2A and H2B h i s t o n e s , all other core h i s t o n -

es are highly c o n s e r v a t i v e in their p r i a a r y s t r u c t u r e (43) and have siailar

n o b i l i t i e s in SDS gels (Figure 1 ) . In rel a t i o n to other p r o p e r t i e s of the

chro a a t i n used in this study, He can ae n t i o n that in h i g h l y t r a n s c r i p t l o n a l -

ly a c t i v e yeast c h r o a a t i n h i s t o n e HI is likely to be absent ( 4 4 ) . T h u is

con s i s t e n t with an earlier o b s e r v a t i o n that even a o o d e r a t e t r a n s c r i p t i o n

leads to the reaoval of HI froo c h r o m a t i n ( 2 4 ) . The repeat length of c h r o a a -

tin d e p e n d s on its ac t i v i t y (being longer in r e p r e s s e d n u c l e i ) and is about

2 4 0 , 2 1 0 , 180 and 165 bp in chroaatin froo sea urchin i p e r c , chicken e r y t h -

3445


r o c y t e s , D r o s o p h i l a e a b r y o s and yeast, r e s p e c t i v e l y ( 1 ) .

E x p e r i a e n t a l a p p r o a c h

To study the n u c l a o s o a e s t r u c t u r e as it exists in n u c l e i , h i s t o n e - D N A

c o n t a c t s were fixed by crosslin k i n g directly in nuclei of sea urchin spera,

yeast and lily. Then the nuclei were digested with a i c r o c o c c a l n u c l e a s e and

the c o r e s were p u r i f i e d either by gel e l e c t r o p h o r e s i s (30) for sea urchin

spera and lily buds or by sucrose gradient c e n t r i f u g a t i o n for yeast. On the

other hind, the ch i c k e n e r y t h r o c y t e and Drosophila e a b r y o c o r e s were c r o s s -

linked after their isolation in order to coepare our re s u l t s with p r e v i o u s

s t u d i e s .

The aethod of locating protein c o n t a c t s on DNA Has d e s c r i b e d in full

detail e l s e w h e r e when used to study the primary o r g a n i z a t i o n of n u c l e o s a a e s

(7) and the RNA p o l y e e r a s e - p r o a o t e r coaplex ( 4 5 ) . The eethod c o n s i s t s of

cro s s l i n k i n g p r o t e i n N H j - g r o u p s to DNA part i a l l y d e p u r i n a t e d under aild

c o n d i t i o n s ( 2 9 ) . The cro s s l i n k i n g causes the DNA to split in such a aanner

that only the 5 - t e r o i n a l DNA fragment b e c o a e s attached to prot e i n a o l e c u l -

es. T h u s , the length of a crosslinked DNA fragaent p r e c i s e l y shows the d i s -

tance of a protein c r o s s l i n k i n g site froa the DNA 5 - e n d . This length can be

assessed by using two systeas of two - d i m e n s i o n a l diagonal gel e l e c t r o p h o r e -

sis. E l e c t r o p h o r e s i s in the first d i r e c t i o n is the saae for the two s y s t e a s ,

with f racti onat l on of cro s s l i n k e d a a t e n a l depending on the size of both DNA

and p r o t e i n s . Then in the first systea of the tw o - d i a e n s i o n a l gel e l e c t r o -

p h o r e s i s , which s e r v e s to identify the crosslinked DNA (DNA-ident ifying s y s -

tei) , h i s t o n e s ara dige s t e d d i r e c t l y in the gel by Pr o n a s e (38) and the r e -

leased " P - l a b e l e d DNA fra g m e n t s are separated in the second d i r e c t i o n

according to their length in th e p r e s e n c e of unlabeled size a a r k e r s (DNA

fra g n e n t s froa D N A a s e I digest of rat liver n u c l e i , see ref. 3 6 ) . The c r o s s -

linked p r o t e i n s d e c r e a s e the DNA aob i l i t y in the first d i r e c t i o n p r o p o r t i o n -

ally to the prot e i n s i z e . As a result, the DNA f r a g a e n t s a t t a c h e d to d i f f e r -

ent h i s t o n e s fall on different d i a g o n a l s in the gel of the second d i r e c t i o n .

All core h i s t o n e s froa sea urchin spera are p a r t i c u l a r l y well resolved in

the first d i r e c t i o n in gels c o n t a i n i n g and n o t - c o n t a i n i n g urea (see F i g u r e

la) and th e r e f o r e the DNA fr a g a e n t s crosslinked to thea are also c o a p l e t e l y

separated into four distinct d i a g o n a l s in the second d i r e c t i o n . T h u s , l o c a -

tion of binding sites for all four core h i s t o n e s along speraal core DNA can

be p r e c i s e l y (* 3-5 n u c l e o t i d e s ) read from p o s i t i o n s of r a d i o a c t i v e spots

on t h e s e d i a g o n a l s (Figures 2 - 3 ) .

To attain a better r e s o l u t i o n and identify the crosslintced speraal DNA

fra g a e n t s of about 6 0 - 1 4 0 , 4 0 - 8 0 and 20 - 4 0 n u c l e o t i d e s long, we used 3 t w o -

diiensional gels of different length and co n c e n t r a t i o n (Figures 2 - 3 ) . For

34 4 6


rH2A

Figure 3. Tno-diaensional gel electrophoresis of "P-labeled DNA cross-linked to histories in speraal corei used to reiolve intermediate and shortDNA fragments.Electrophoresis in the -first direction Has carried out (A) in 151 polyac-rylaaide slab gel (165x365x0.6 ••) and (B) in 1BX polyacrylaaide slab gel(165x165x0.6 ••) at a conitant current of 3 aA and 7 aA -for the concentrat-ing and separating gel, respectively.Electrophoresis in the second direction Has performed (A) in 151 polyacryl-aaide slab gel (200x400x1 ••) and (B) in 30Z polyacrylaaide slab gel (200xx200xl ••) at 7 aA and 14 BA -for the concentrating and separating gel, res-pectively. For other details see Figure 2.

lily nucleosoaal cores, this systea did not separate the diagonals

of DNA fragaents attached to histone H3 and to the aain H2A fraction. In

this case, identification of the H2A-bound DNA fragaents Has carried out

using a very weak H2A-subfraction diagonal which Has detected between the H3

and H4 diagonals indicated in Figure 4.

For the cores froa any other source, the diagonals for H2A- and H2B-cross-

1 inked DNA fragaents Here separated rather poorly in the DNA-identifying two-

diaensional systea. Then He used a second systea of two-diaensionil gel elec-

trophoresis which identified the crosslinked proteins (protein-identifying

3447


6 0 -

Figure 4. Tno-diaensional gel of" P - l a b e l e d DNA froa lily budcrosslinked cores.Exper1nental parameters Mere thesaae as in Figure 2.

systeo) and provided an lcproved resolution -for the crosslinked histones

froa Drosophi1 a, yeast and chicken erythrocytes: following fractionation

of crosslinked material in the first direction, the DNfl Has hydrolysed d i -

rectly in the gel, and the released * a o I - l a b e l e d histones Here then sepa-

rated in the second direction. In this gel (Figure 5 ) , the spots o-f cr o s s -

linked histones are shifted to the le-ft froa the spots of uncrossl inked ones

(the extreae right part of the autoradiograa) . By coaparing the protein-

and DNA-identifying gels (Figures 5 - 6 ) , one can accurately deteraina the DNA

length. Here He have coapared such gels for chicken e r y t h r o c y t e cores only;

the coaparison for Drosophila cores Has done earlier (7) and the saae for

yeast and lily buds is to be published elsewhere.

A better resolution of tno-dieensional gels here as coapared with pre-

viously published results (7) his been attained owing to the reaoval of

oost uncrossl inked DNA froa the crosslinked a a t e n a l on a hydroxylapatite

coluan prior to DNA-identifying two-di«ensional gel el e c t r o p h o r e s i s , and

also because m used thinner and longer gels, gsl con c s n t r a t i o n s and cur-

rent Here optisized, etc.

Arrangement of histo n t s on DNfl

The data on the arrangement of histones on DNA in nuclsosoaal cores sua-

narizing 3-5 e x p e r i m e n t s , such as presented in Figures 2-6, are shown for

chicken e r y t h r o c y t e s , Drosophi 1 a sabryos and yeast particles in Figure 7A,

for speroal cores in Fig u r s 7B and for lily particles in Figure 7C. Also g i -

ven are rel a t i v e intensities of histone crosslinking to different DNA seg-

3 4 4 8


e e n t s . Here we have di s e n 01 nated for the first tiae the binding sites

between histones H2A and H2B in the area of 110-130 n u c l e o t i d e s and be t w e e n

H2A and H3 within 130-145 n u c l e o t i d e s froa the 5' DNft t e r a i n i . The p r e v i o u -

sly published data (7) are sup p l e o e n t e d with newly d i s c o v e r e d weak c r o i s l i n -

king sites for all h i s t o n e s . He have also found a direct c o r r e l a t i o n between

the a p p e a r a n c e of the cr o s s l i n k i n g sites H 4 4 O l T . and the p r e s e n c e of par t -

icles containing about 135 bp long DNA in the core p r e p a r a t i o n s (in pure

nucleosoaal c o r e s these c r o s s l i n k i n g s i t e s were not o b s e r v e d ) . He do

not know the reason for the horizontal twinning of the H 3 T O . » O spots in

the DNA gels of spernal cores (Figure 2 ) . This lay hav e resulted froe p a r t i -

al aodification or p r o t e o l y s i s of H3 . The vertical doubling of the H 4 B B , * O

s p ats in the gels of speraal corei on the saae figure seeai to be due to an

incoaplete digestion of this h i s t o n e by Pronase before e l e c t r o p h o r e s i s in

the second d i r e c t i o n .

Froa Figures 7 and 8 it is apparent that all the cores studied have very

sieilar arrangeaent of his t o n e c r o s s l i n k i n g sites along DNA, even though the

rel a t i v e intensity of c r o s s l i n k i n g can differ at a pa r t i c u l a r s i t e . Only the

strong c r o s s l i n k i n g site H 2 B O o and the weak site H 3 1 O o ihow ioae s p e c i -

ficity. Thete sites ware found only in the cores froa sea urchin spera and

lily buds. It seeas that the additional cluster of 4 to 5 lysine r e s i d u e s in

the extended N-terainal part of speraal H2B (41) is involved in the H 2 B O «

c o n t a c t . He h*v» to adait here that the H2B crosilinking in the H 2 B , 3 _ O ,

area s i g n i f i c a n t l y varied in intensity and relative s h a r p n e s s of spots in

two-dimen s i o n a l gels, even when the e x p e n a e n t s were done with the saae core

p r e p a r a t i o n s . This p r o b a b l y r e f l e c t s a high s e n s i t i v i t y of c r o s s l i n k i n g to

the experimental c o n d i t i o n s owing to an oscillation of H2B between the com-

p l e m e n t a r y DNA st r a n d s a c r o s s the DNA gr o o v e s (7) and between the a d j a c e n t

turns of superhelical core DNA (see F i g u r e 9) which aay account for the H2B

crosslinking in this area. Less p r o n o u n c e d , though still n o t i c e a b l t , v a r i -

a t i o n s in intensity were also observed for crosslinking of other h i s t o n e s .

Another d i s s i o i I a n t y , a l r e a d y recorded earlier ( 8 ) , is the fact that

the H 2 A 7 s site is found only in the cores crosslinked after their isola-

tion, e. g. in chicken and D r o s o p h i l a p a r t i c l e s . This is prob a b l y an in-

dication that the isolation p r o c e d u r e a f f e c t s the core s t r u c t u r e .

DISCUSSION

Structural i m p l i c a t i o n s

He have not found any essential d i f f s r s n c a i in the primary o r g a n i z a t i o n

of n u d e o s o n a l core p a r t i c l e s o r i g i n a t i n g froa all three higher e u k a r y o t e

kingdoos (aniaals, p l a n t s and f u n g i ) ( 5 1 ) , naaaly froa sea urchin spire (Fi-

3 4 4 9


143 958575

35

88 65 55

H3143 95 8575 68 58

H2A143-118 / /

115 105 95 48 35 25

^ H 2 B

H2A I—I143

A I I143 118 35

H498 88 65 55 45

3450


Figure 5. Two-diaensional gel el ectrophoresis of 1 1 BI-labeled historiescrosslinked to DNA in Drosophila eabryo (ft), yeast (B) and chicken eryth-rocyte (C, D) cores.The crosslinked coaplex Has electrophoresed in the first direction in 15Xpolyacrylaaide slab gel (200x200x0.6 ••) containing 7 PI urea at 3 aA and 7• A for the concentrating and separating gel, respectively. Folloning hydro-lysis of DNA in the gel, electrophoresis in the second direction Has per-foraed in 15X polyacrylaaide slab gel (200x400x1 aa) at 3 aA and 5 aA forthe concentrating and separating gel, respectively. Electrophoresis Hasstopped when broaphenol blue reached the bottoa of the gel. The spots ofuncrosslinked histones are se«n at the extreae right of the gel. The figureat each spot indicates the size of DNA fragaents crosslinked to h u t o n e s asdeterained froa Figure 6. (D) A longer exposure of the gel shonn in (C). X-unidentified spot.

gure 7A), lily buds (Figure 7C), Drosophila eabryos, Ehrlich ascite aouse

cells and rat liver (the latter three Here studied earlier in this laborato-

ry, ref. 7 ) , and yeast (Figure 7A). The fen exceptions Here the strong con-

tact H2B O. in sea urchin spera, the relatively weak contact H3 1 Oo in so-

aatic cells of lily and the H 2 A 7 B contact found only in corti croislinked

after isolation. These data further support the idea that the core nucleo-

soae structure is highly conserved in evolution (1).

Figure SA suaaarizes data on the arrangement of aain histone contacts

along one strand of core DNA. Figures SB and 1 shon, respectively, aodels

for the symmetrical arrangement of histones on the double-stranded linear

and superhelical DNA. It can be seen that histones are aligned on one core

DNA strand in the following order:

(51) H2B2B,3B-H4BB,*0-H37a(aa,vo/H4aa-H2Bioa,iia~H2Aiia-H3ixB,i4B/H2Ai4B (3').

On the double-stranded core DNA the order of histones is as follows:

H2A/H3-(H2A-H2B)-(H3,H4) 2-(H2B-H2A)-H3/H2A.

The weak contacts of histones with core DNA which are absent in Figures

B,A-B tre shown in Figures 7,A-C. These weak contacts eight arise ts a rt-

sult of siaultaneous interaction of histone aoleculas and their lysine re-

3451


Figure 6. T n o - d m e n s i o n a l gel of " P - l a b e l e d DMA from chicken eryth-rocyte crosslinked cores.For experimental paraaeters iee Figure 2. (ft) The top part of the gel aftera short exposure; (B) the whole gel after a longer exposure.

sidues Kith both complementary DNA strands (7) or Hith the adjacent super-

helical turns of core DNA. They could also be accounted for by small

amounts of various nucleosoie conforaers and other particles prtsent in

the preparations of core nucleosoaes. It is hoped that the naturi of the

weak contacts may be better understood by identifying regions in histone

molecules that actually inttract Hith each particular DNA segment.

The additional contact H 2 B S S which is characteristic of sea urchin

spirm cores most probably appears because the N-ttrminal region of jpereal

H2B contains 4 to 3 additional lysine residues. The additional interaction

site of the spernal H2B with DNA say stabilize its binding to the other DNA

segments and thus give a better protection against DNAase I attack to the

sites localized at a distance of 20, 40 and 50 bases from the S'-and of the

core DNA and increase thermoitabi1ity of these core particles (12). It is

interesting that in contrast to a higher stability of sea urchin sperm nuc-

leosooal cores as conpared with chicken erythrocyte corts (12), yeast c o r n

possess a relatively sore relaxed structure than erythrocyte coras (14).

No crosslinking or very iieak crosslinking of histones Hith the first 20

bases, in the regions of 40-50 and 122-130 bases from the 5'end of core DNA

(Figures 7, A-B and 8A; see ilso ref. 10) can be correlated with some ear-

lier observations, namelyi the Ion thermostabi1ity of the first 20 bases

3452


H2AH28

O » JO 1O «O bO *O 70 tO * 0 WO <<0 <H ' »0 «4£

c

H28 a i aai m • • _ aai aai aai _H3 - - °"D c = ™

1 I- X ± L X X X X X J_ L, L, l_ L_O to ?O JO *O 50 tO W tO 9O <OO IIO IJO^ i»O »*4

H2A - - a i "H26 _ cz> aai aai _

H 3 _ _ ciaia — C = B »

H4 — aai aa ._ aai _l i i j i j I x ' ' I ' l l 'o t o » 5 3 4 0 5 5 * o T O B o 9o noiio3oijoSo

Figure 7. Arrangement of histones on DNA in the nucleosoaal core particles.Location of histone crosslinking sites on one DNA strand is shown for chick-en erythrocyte and Drosophila eebryo cores crosslinked after isolation andyeast cores crosslinked in nuclei ( A ) , and -for sea urchin speraal (B) andlily (C) cores crosslinked within nuclei. The crosslinking efficiencies wereestinated froa radioactivity of corresponding spots in two-diaensional DNA-and protein-identifying gels and are shown in the order of 3-5 fold d e c r e a s -ing by black bars, open bars, solid lines, and broken lines. Distances alongDNA are indicated in nucleotides froa the 5'-tnds (the true values are lis-ted for Figure 2 ) .

of core DNA (52, 5 3 ) ; the presence of very few histone-DNA ionic bonds in

this region ( 5 4 ) ; the existence of coapact dinucleosoaes foraed by overlap-

ping of the adjacent core particles at their terainal regions (55), and the

localization of sites preferentially accessible to DNAase I (Figure 8A) at

distances of 10, 20, 40, 50, 120 and 130 bases froa the 5'-end of core DNA

(12, 4 8 - 5 0 ) . It should be pointed out that soae of the aost accessible sites

are situated between the regions of diner (H2A-H2B) and tetraaer ( H 3 , H 4 ) ,

crosslinking.

The histone octaaer of a nucleosoaal core particle consists of relative-

ly autonoaous, although interacting with each other, specific histone comp-

lexes: one tetraaer (H3, H 4 ) x and two diaers (H2A-H2B) ( 1 ) . In core nuc-

leosoaes, the tetraaer and the two diners are coaparatively independent! the

tetraaer is located in the center of the core DNA froa -2.5 to +2.5 sites

(Figures 8B and 9) while the two diners flank tht tetraaer on both sides and

bind to both ends of the core DNA. This aodel was supported in a study of

the subnucleosoae structure ( 5 6 ) . On the other hand, within DNA areas acco-

aodating the (H2A-H2B) diaers and, in particular, the ( H 3 , H 4 ) 2 tetraaer

there is a significant overlapping between h i s t o n t s . Siaultaneous interacti-

3 4 5 3


M M I I M3i>O

BMl' H?rf HZjl

"2B' M2B' H4 Mi M ] 1 M T M T M2B' M?Bf "31 HJ1

3

0 10

146 UO

-7 -

20

130

t -5

50

120 1

-4

40

10

-3

— t —50

100

•2

60

90

H 4'

-1

70

eo

/ eo

0

90

60

1

100

50

2

110

40

3

120

30

4

—t—130

20

5

—t-140

10

-1146

0

7

Figure 8. (A) A lap for the main histone contacts located on one DNA strandwhich tuiiarizei results of the crosslinking studies on cores froe chickenerythrocytes, Drosophila eibryoa, yeast, sea urchin »per» and lily buds(Figures 7 A,B,C) and earlier studied cores -fro« rat liver and I O U S I ascitetu«or cells (7, 8 ) . Specific interaction with DNA of H2B froo sea urchinspera is not shown in this »ap. The contact H 2 A 7 B (in brackets) was foundonly in tha cores crosslinked after isolation. The arrows point to the laincore DNA sites exposed to DNAase I, the arrow length being proportional totht relative accessibility (12, 49, 5 0 ) .(B) A symmetrical aodel for the arrangement of histones on two DNA strandsin the core particle. The eodel is based on the data of Figure BA and assuo-es the presence of a dyad axis of symmetry in the cores ( 4 ) . Superscripts 1and 2 denote two copies of each histone in tht core. Distances along DNAare given either in nucleotides from the 5'-end or by numbers from -7 to +7taking the diad symmetry axis in the core particle as the origin, to mark14 r e p e a t s in the 145 bp long double helical core DNA ( 4 ) .

H2B± --

Figure 9. A three-diaensional aodel for the symmetrical arrangement of his-tones on the folded core DNA.The DNA forms a left-handed superhelix containing about 80 bp par turn (3).Positioning of histones H2A (narrow box, broken line), H2B (wide box, bro-ken line), H3 (wide box, continuous line), and H4 (narrow box, continuousline) on two DNA strands is shown by placing histones above and below theDNA line and is taken froa Figure B.

3454


on of histone H3 with the central ( H J T B . O O . V B ) and terainal ( H 3 , 3 H l l « o )

regions of the core DNA located close to each other on the folded DNA (Figu-

re 9) seems to stabilize the DNA superhelix (4, 7 ) . This is consistent

Nith the finding that just one (H 3 , H 4 ) a tetraaer suffices to iike the core

DNA fold (57).

The fact that the new contact H2A To appears only in n o l a t e d cores

brings forth the question of its genesis. This contact aay appear as a re-

sult of soae rearrangeaent of histone H2A on DNA induced by thi absence of

either HI and spacer DNA or lnternuc1eosooal contacts in isolated cores.

Our preliminary data have also demonstrated the absence of the H2A 7 s con-

tact in isolated Hl-containing nucleosoaes crosilinked after preparation,

where H2A was not crosslinked to the spacer DNA. This suggests that H2A can

be involved in internucieosoaal contacts. Participation of core histonei in

interactions between nucleosoaes in chroaatin was proposed earlier (2, 5 8 ) .

It is of interest to coapare our high resolution aap for the arrange-

aent of histones on DNA in the cores (Figures 8 and 9) with the aodel of

three-dmensional structure of the cores based on recent X-ray and neutron

diffraction studies of core crystals by Richaond et al . (6) and Bentley et

al. (5). Both approaches have led to a conclusion that histones are aligned

along the whole DNA length with histone-DNA interactions occurring on the

inside of the DNA superhelix. The si and ̂ 4 sites of DNA sharp bending

(6) correspond to the region of siaultaneous interaction of H3/H4 and

H2A/H2B with DNA at 85/65 and 115/35 nucleotide* froa the 5' DNA ends. The

slightly different arrangecent of two (H2A-H2B) diaers on DNA and m v o l v e -

aent of H2A in the intercore contacts in the core crystals suggested by

Richaond et <1. (6) agree with our data on the easy rearrangenent of H2A

(found by coaparing isolated cores and cores within chroaatin) and with the

variable pattern of H2B crosslinking in the area of 20-50 nucleotides froa

the 3' DNA ends. On the other hand, the assigneaent of the C-rod-like

structure in the region froa -3.5 to 1 5 to histone H4 is not supported

by our data since we observed H2A and H2B but no H4 crosslinking within

these DNA segaents. The X-ray, neutron diffraction and crosslinking data

have been recently supported by direct investigations of topography of his-

tone-DNA interactions in core particles reconstituted froa individual histo-

nes, labeled by P t * M 5 9 ) . Other details of the core structure will hope-

fully be revealed when further inproveaents in resolution are complemented

with identification of the regions in histone aolecules that art crosslinked

to each particular segaent of core DNA.

The ten-nucleotide periodicity in the arrangeaent of hittone crosslinking

sites on core DNA (see Figures 7 and 8 in ref. 10) is auch sooner deterained

3455


by the lateral arrangement of histones on the internal side of DNA in the

n u d e o s o a e than by any specificity in DNA aethylation and digestion with

•icrococcal nuclease (60). In our aock experiments we have found no regula-

rity in crosil inki ng lysines instead of histones to core DNA (not shoiin).

Functional iaplications

The sieilarity of the linear arrangement of histones in nucleosoaal cores

isolated either from the repressed nuclei (sea urchin sperm or chicken eryt-

hrocytes) or from the nuclei that ire actively involved in transcription and

replication (Drosophila embryos and especially yeast) suggests that the

overall inactivation of chromatin cannot be reflected and/or regulated at

this primary level of chroeatin organization.

Me do not exclude, however, that there can bi othtr structural features

in active nucleosomes that have remained undetected by the methods described

above. Such processes <s histone modification, HM6-protein binding, partial

unfolding, etc., might affect histone-histone interactions but not the loca-

tion of their contacts on DNA. It has been shown that the unfolding of core

particles which occurs at high concentrations of urea and NaCl (61) and the

binding of two HMG 14/17 molecules to the cores and Hl-containing nucleosom-

es (62) do not lead to any substantial changes in their primary organization.

The low conforaatlonal stability of yeast nucleosoaes (14) is not reflected

in their primary organization either. However, all these factors may facili-

tate reaoval of histones froa transcribed DNA (23, 2 4 , 6 3 ) .

Specific features of both active and repressed chroaatin may reveal them-

selves on higher levels of chrooatin structure. Thus, the removal of histone

HI from moderately transcribed heat-shock Droaophila genes, observed in

our laboratory (24) seems to induce unfolding of the 30-nm chromatin fiber

into the 10-ne fiber. The presence of spereal variants of histone H2B in sta

urchin sperm chroaatin hinder such transitions as well as the teopo-

rary raooval of histone octamers from the sites of transciption and thereby

inhibit transcription. The speraal variants of H2B increase the t m d e n c y of

nucleosoaes to aggregate (26) and participate in the organization of linker

DNA ( 6 4 ) , thus stabilizing the condensed structure of repressed speraal

chroaatin. Studying the presence of histones in increasingly activated heat

shock genes of Drosophila, hsp 22 and hsp 70, it was shown that all HI and

some core histones are reaoved upon aodarate transcription while upon active

transcription the gene DNA becomes completely fres of histones (24, 6 3 ) .

Considering the aechaniss by which RNA polyaerases could displace histones

froa DNA and affect the n u d e o s o m e structure, one has to take into account

the essentially nonoverlapping arrangement of the ( H 3 , H 4 ) , tetraaer and

3456


the (H2A-H2B) diaers on the central and terminal reqioni of core DNA and

siaultaneous interaction of histone H3 with the both region* in folded cor-

es (Figures 8 and 9 ) . Entering the terminal region of core DNft, RNA po l y a e -

rase first displaces histone H3 and then one (H2A-H2B) diner. These displa-

cements will unravel the tercinal DNA region or even the whole nucleosoae,

for exaaple in such a way as was observed in Physarua n b o s o a a l chroaatin

by Prior et al. ( 2 2 ) . Upon its further aove, the polyaerase will reaove the

tetraaer and finally the second hiitone diaer. Consistent with this nodal

is the finding by Baer and Rhodes (20) that the RNA polyaerase-nucleosoaal

core coaplex is deficient in one (H2A-H2B) d n e r . ThB low extent of over-

lapping between the histone tetraaer and two diners on core DNA agrees wall

with their independent segregation during replication of chroaatin ( 6 5 - 6 9 ) .

ACKNOHLEDBEHENT

The authori are grateful to D . P r o m s for help in so«e of the exper i n e n t s ,

to V.Korobko for the gift of polyn u c l e o t i d e kinase, to K.Ebralidze and

V.Karpov for stieulating discussions and to T.Richaond who acquainted us

with his paper before its publication.

*To whom correspondence should be addressed

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Documents

volume 13 Number 10 1985 Nucleic Acids Research · Nucleic Acids Research cation, in the presence of histone variants and nonhistone proteins (for re-viex see 1, 2, 11) and in soae