volume 5 Number 7 July 1978
Nucleic Acids Research
Alteration in nucleosome structure induced by thermal denaturation
V.L.Seligy and N.H.Poon
Molecular Genetics Group, Division of Biological Sciences, National Research Council, Ottawa,
K1A0R6 Canada
Received 19 May 1978
ABSTRACT
Mononucl e o s o m e s prepared from goose e r y t h r o c y t e nuclei
exhibited limited heterogeneity with respect to number o f
e l e c t r o p h o r e t i c c o m p o n e n t s , h i s t o n e s and DNA c o m p o s i t i o n . T h e
c o m p o n e n t s d i f f e r s l i g h t l y in i o n i c s t r e n g t h i n d u c e d s e l f a s s o c i a t i o n . T h e r m a l d e n a t u r a t i o n o f e a c h c o m p o n e n t g a v e only
two d o m i n a n t , h i g h l y c o o p e r a t i v e , m e l t i n g t r a n s i t i o n s , T " and
T " ' . U r e a and t r y p s i n w e r e used t o e s t a b l i s h the d i f f e r e n t i a l
l a b i l i t y o f t h e s e two t r a n s i t i o n s . C o m p a r i s o n o f the m o r p h o l o g i e s
of the m o n o n u c l e o s o m e s a t v a r i o u s s t a g e s t h r o u g h o u t the m e l t i n g
p r o f i l e i n d i c a t e d t h a t the 13.3 ± 1.5 n m d i a m e t e r m o n o n u c l e o s o m e s
s t a r t t o d i s r u p t o n l y in the l a t t e r h a l f o f t r a n s i t i o n T " and d o
not u n f o l d until a f t e r r e a c h i n g T " ' . T h e r e s u l t a n t , o p e n ended
( 1 7 . 4 ± 2.2 n m d i a m e t e r ) t o r o i d s a r e s t i l l l a r g e l y n e g a t i v e l y
s t a i n i n g and m u c h m o r e u n i f o r m in s h a p e i f f i x e d s i m u l t a n e o u s l y
with gluteraldehyde.
INTRODUCTION
E l e c t r o n m i c r o s c o p y and t h e r m a l d e n a t u r a t i o n a r e two o f
m a n y t e c h n i q u e s w h i c h h a v e been used e x t e n s i v e l y to p r o b e c h r o m a tin and its m a j o r s u b s t r u c t u r a l c o m p o n e n t , the n u c l e o s o m e (1) o r
v - b o d y ( 2 ) . T h e l a t t e r has been e x t e n s i v e l y c h a r a c t e r i z e d by
p h y s i c a l and b i o c h e m i c a l m e t h o d s (see c u r r e n t r e v i e w s 3 - 6 ) and
by e l e c t r o n m i c r o s c o p y u s i n g b r i g h t a n d d a r k f i e l d m o d e s
( 1 , 2 , 6 - 1 5 ) . A large number of attempts have been made to interp r e t the s i g n i f i c a n c e o f the t h e r m a l m e l t i n g t r a n s i t i o n s o f
c h r o m a t i n and n u c l e o s o m e s w i t h r e s p e c t t o s p e c i f i c i n t e r a c t i o n s
of the DNA and p r o t e i n m o i e t i e s ( 1 6 - 3 5 ) . V i s u a l i z a t i o n o f an
a l t e r e d n u c l e o s o m e s t r u c t u r e , such a s f o u n d in the c a s e for u r e a
d e n a t u r e d n u c l e o s o m e s ( 2 7 , 3 6 ) , m a y b e v e r y u s e f u l for f i n a l
interpretation.
In the p r e s e n t s t u d y w e c o r r e l a t e d v i s u a l c h a n g e s in
© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
Nucleic Acids Research
n u c l e o s o m a l m o r p h o l o g y w i t h the v a r i o u s t h e r m a l t r a n s i t i o n s o f
the DNA a s s o c i a t e d w i t h n u c l e o s o m e s . C o n t r o l l e d , t h e r m a l l y
d e n a t u r e d m o n o n u c l e o s o m e s u n f o l d to y i e l d i m a g e s s i m i l a r to
those described for urea denatured nucleosomes ( 2 7 , 3 6 ) .
The
d a t a a r e c o n s i s t e n t w i t h a n a r r a n g e m e n t o f D N A s i m i l a r to t h a t
r e c e n t l y p r e d i c t e d from neutron scattering and X-ray d i f f r a c t i o n
observations (3,6,15,37).
MATERIALS AND METHODS
Preparation of n u c l e o s o m e s : Digests of intact goose erythrocytes
(14) or chromatin (38) were carried out with micrococcal nuclease
(E.C. 3.1.4.7, code NFCP, Worthington Biochemical Corp., actual
a s s a y 2 6 . 8 u n i t s p e r m i c r o g r a m ) at 2 3 ° . T h e e n z y m e w a s a s s a y e d
f o r h i s t o n e p r o t e o l y t i c a c t i v i t y and f o u n d to be nil up to 24 hrs
at 3 7 ° . Nuclear digests were terminated after about 1 5 % acid
s o l u b i l i t y by a d d i t i o n o f t h r e e v o l u m e s o f i c e c o l d 2 m M E D T A pH
8 . 0 . T h e d i l u t e d d i g e s t s , a b o u t 0.05 in i o n i c s t r e n g t h f o r n u c l e i ,
w e r e g e n t l y s t i r r e d w i t h a m i c r o m a g n e t i c b a r f o r 5 m i n u t e s a t 0°C
b e f o r e e n r i c h i n g f o r c h r o m a t i n f r a g m e n t s ( r a n g e 3s < 5 0 s ) by
c e n t r i f u g a t i o n f o r 15 m i n u t e s a t 2 0 0 0 xg in a s w i n g i n g b u c k e t
r o t o r . C h r o m a t i n f r a g m e n t s c o r r e s p o n d i n g to b r i g h t f i e l d e l e c t r o n
microscopically defined nucleosomes (7-11,14) were further isol a t e d by u l t r a c e n t r i f u g a t i o n ( 3 9 ) and ul t r a f i 1 t r a t i o n ( 1 4 ) . T h e
r e l a t i v e purity of the nucleoprotein preparations was assessed
b y c o m p a r i s o n o f e l e c t r o p h o r e t i c m o b i l i t i e s w i t h nucl e o p r o t e i n s
from the o r i g i n a l d i g e s t and DNA e x t r a c t e d from the p u r e m o n o n u c l e o s o m e f r a c t i o n s ( 1 4 , 4 0 , 4 1 ) . R e f e r e n c e DNA m a r k e r s w e r e from
X p h a g e a n d P M 2 D N A d i g e s t e d w i t h Hpa II a n d H a e I I I , r e s p e c t i v e l y .
P r o t e i n s w e r e a n a l y z e d on 1 2 % and 2 0 % p o l y a c r y l a m i d e gels
( 4 2 , 4 3 ) u s i n g p u r i f i e d h i s t o n e s and c r o s s - l i n k e d m a r k e r s , 1 4 , 3 0 0
t o 7 1 , 5 0 0 MW ( 8 D H B i o c h e m i c a l s L t d . , P o o l e E n g l a n d ) .
Pure l y o p h i l i z e d h i s t o n e s consisting of H 2 A , H 2 B , H3 and
H 4 ( 1 : 1 : 1 : 1 ) all f r o m m a t u r e g o o s e e r y t h r o c y t e s ( 4 4 ) w e r e w e i g h e d
o u t , d i s s o l v e d in g l a s s d i s t i l l e d w a t e r a n d a d j u s t e d to pH 7.0
w i t h a d d i t i o n o f s o d i u m p h o s p h a t e to 5 m M . T h e p r o t e i n s t o c k w a s
2.5 m g / m l .
Thermal denaturation:
S a m p l e s w e r e a n a l y z e d by c o m p a r i n g the
m e l t i n g c u r v e s a n d f i r s t d e r i v a t i v e p l o t s o b t a i n e d in 5 m M N a P O .
pH 7 . 0 . T h e r m a l d e n a t u r a t i o n w a s d o n e in a m o d i f i e d G i l f o r d 2 4 0
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e q u i p p e d w i t h a 6 0 4 6 a n a l o g m u l t i p l e x e r and 2 5 2 7 t h e r m o p r o g r a m m e r . The t h e r m o c u v e t t e assembly was e l e c t r o n i c a l l y heated.
The m e t h o d for d i g i t a l i z i n g the data and c o m p u t e r a n a l y s i s h a s
been briefly described ( 2 4 ) . For thermal d e n a t u r a t i o n studies
i n v o l v i n g e l e c t r o n m i c r o s c o p i c a n a l y s i s , the s a m p l e s w e r e h e a t e d
in a m a n n e r e q u i v a l e n t t o t h e 2 5 2 7 p r o g r a m m e r s y s t e m .
Specific
e x p e r i m e n t a l d e t a i l s a r e g i v e n in f i g u r e d e s c r i p t i o n s .
Electron microscopy:
For routine e l e c t r o n m i c r o s c o p i c work
aliquots o f unfixed o r g l u t e r a l d e h y d e fixed n u c l e o s o m e s , h i s tones o r DNA were placed o n Kodak P h o t o - f l o ( 0 . 1 % v/v) p r e w a s h e d o r UV (254 n m ) i r r a d i a t e d f o r m v a r coated grids (14) an
e q u a l v o l u m e o f e i t h e r uranyl a c e t a t e s t a i n o r 4 % ( v / v ) g l u t a r a l d e h y d e . The sample grids were subsequently flushed with filtered
d i s t i l l e d w a t e r , s t a i n e d (in t h e c a s e o f f i x e d s a m p l e s ) , a i r
d r i e d , a n d sandwiched between the formvar with a c o a t i n g o f
e v a p o r a t e d carbon (45) a t 5 x 1 0 m m Hg. The n e g a t i v e stain
was u s u a l l y 1% (w/v) uranyl a c e t a t e - 5 % a c e t o n e ; v a r i a t i o n s
over the range o f 0 . 0 0 5 % to 5 % a q u e o u s uranyl a c e t a t e w e r e
examined. All images were recorded on Kodak Electron Image
Plates using a Seimens Elmiskop 1A at 80KV. Optical e n l a r g e m e n t o f the original i m a g e s was d e s c r i b e d p r e v i o u s l y using t h e
M a c r o s y s t e m and c o n v e n t i o n a l e n l a r g e r ( 1 4 ) . N o c h r o m a t i c a b b e r a tions o r image d i s t o r t i o n s were detected. The size o f an o b j e c t
was determined from m e a s u r e m e n t s either from accurately enlarged
p r i n t s , using neutral contrast p a p e r , o r as we p r e f e r r e d , from
the original negatives viewed by an optical m i c r o s c o p e and an
eye piece with a calibrated s c a l e . The latter method is considered more precise b e c a u s e the original plates c o n t a i n i n g t h e
p r i m a r y e l e c t r o n i m a g e s a r e i m m u n e t o m e a s u r e m e n t l o s s in c o m p a r i s o n t o t h e e n l a r g e d p r i n t s w h i c h c a n r e s u l t in a s m u c h a s
1 0 % d e v i a t i o n a s the print c o n t r a s t is i n c r e a s e d .
Determination o f temperature discrepancies between actual
sample heating t e m p e r a t u r e and sample t e m p e r a t u r e a t i n c i p i e n t
transfer to electron m i c r o s c o p e grids was facilitated b y use o f
custom made microthermocouples which were geometrically fixed
into five microlitre p i p e t t e s . The exact temperature and durat i o n in s e c o n d s w e r e r e c o r d e d s i m u l t a n e o u s l y u s i n g a M o d e l 7 0 0 1
AM M o s e l y A u t o g r a p h X - Y r e c o r d e r ( H e w l e t t - P a c k a r d ) e q u i p p e d with
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a Bio E 330 pen position adaptor; X-axis sweep was 0.25 seconds
p e r c m ; Y - a x i s t e m p e r a t u r e of e x p a n s i o n w a s 2 . 1 3 ° C p e r c m . F o r
grid temperature measurements, m i c r o t h e r m o c o u p l e probes were
g e o m e t r i c a l l y f i x e d a t 0 . 5 mm a b o v e t h e c e n t e r o f e a c h g r i d .
T h i s d i s t a n c e a l l o w e d f o r full c o n t a c t w i t h f i v e m i c r o l i t r e o r
g r e a t e r b u b b l e s of f i x a t i v e , stain o r w a t e r placed on the g r i d s
prior t o a p p l i c a t i o n of sample. T h e p r o c e d u r e w a s used to avoid
d i r e c t c o n t a c t w i t h t h e p r o b e a n d g r i d a s s e m b l y . T h e g r i d s , in
t u r n , w e r e r i g i d l y h e l d in p o s i t i o n by t h e i r r i m s ( = 0 . 5 m m f r o m
o u t e r e d g e ) w i t h D u m o n t N o . 5, s u p e r - f i n e , l o c k i n g t w e e z e r s .
Results
C h a r a c t e r i z a t i o n o f n u c l e o s o m e s : M o n o n u c l e o s o m e s used f o r
t h e r m a l a n d e l e c t r o n m i c r o s c o p y s t u d i e s a r e m o r e l i m i t e d in
heterogeneity than recently reported (14,28,46-49). Densitom e t r i c scans gave only two major c o m p o n e n t s (Fig. l a ) equivalent
in m o b i l i t y to a b o u t 1 9 4 , 7 0 0 t o 2 4 7 , 5 0 0 d a l t o n s o f D N A ( c a l i brated with H a e III PM2-DNA restriction fragments F to N (Fig.lc)
as a s s i g n e d in r e f . 5 0 ) . A w e i g h t a v e r a g e m o l e c u l a r w e i g h t o f
9 8 , 3 4 0 daltons w a s computed for the DNA (Fig. l b ) . Each component contains histones H 2 A , H 2 B , H3 and H 4 . The dominant species,
about 8 8 % of t h e s a m p l e , c o r r e s p o n d s to t h e "core p a r t i c l e " by
d e f i n i t i o n o f DNA base pair number ( 3 , 4 , 6 ) . We earlier noted
( 1 4 ) t h a t m o n o n u c l e o s o m e s , o b t a i n e d by d i g e s t i o n o f n u c l e i h e l d
i n t a c t by p h y s i o l o g i c a l s a l i n e b u f f e r , w e r e l e s s h e t e r o g e n e o u s
t h a n f r o m n u c l e i o r c h r o m a t i n d i g e s t e d in l o w i o n i c s t r e n g t h
b u f f e r . Since the DNA digestion patterns are about the same
( 2 3 , 3 8 , 5 1 ) t h e h e t e r o g e n e i t y w e o b s e r v e d m a y be r e l a t e d t o
d i f f e r e n c e s in s e l f - a s s o c i a t i o n o f n u c l e o s o m e s i n d u c e d by i n creased ionic strength ( 4 6 ) . We examined this possibility using
c h r o m a t i n d i g e s t e d at l o w ionic s t r e n g t h a n d purified by sucrose
g r a d i e n t c e n t r i f u g a t i o n using l o w and high ionic strength
( 1 4 , 3 9 ) b u f f e r . Figure 2A shows t h e proportion of chromatin
f r a g m e n t s o r p u r i f i e d n u c l e o s o m e s w h i c h d o n o t a g g r e g a t e at t h e
v a r i o u s i o n i c s t r e n g t h s , r e l a t i v e t o 1 5 0 m M NaCl n u c l e o s o m e s . T h e
s o l u b i l i t y o f t h e l o w i o n i c s t r e n g t h d e r i v e d n u c l e o s o m e s is
s i m i l a r t o t h a t o b s e r v e d b y 01 ins et al ( 4 6 ) a n d W i t t i g a n d
Wittig (32). Electrophoresis of the various precipitates and
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F i g u r e 1 . C h a r a c t e r i z a t i o n o f m o n o n u c l e o s o m e s used in c o m b i n e d
t h e r m a l d e n a t u r a t i o n and e l e c t r o n m i c r o s c o p y s t u d i e s . D e n s i t o metric scans (580nm) of photographic negatives of ethidium bromide stained polyacrylamide slabs (0.3cm x 20cm) were made: ( a )
m o n o n u c l e o s o m e s (125 pg D N A ) , (b) DNA f r o m n o m o n u c l e o s o m e s (50 yg]
and (c) Hae III r e s t r i c t i o n f r a g m e n t s o f PM2 DNA (15 p g , 3X c h a r t
e x p a n s i o n ) . The 4 % ( w / v ) p o l y a c r y l a m i d e gel s l a b s w e r e e l e c t r o p h o r e s e d w i t h c o n t i n u o u s b u f f e r c i r c u l a t i o n . C a l i b r a t i o n o f res t r i c t i o n f r a g m e n t s w a s based upon p r e - d e s i g n a t e d s i z e s ( 5 0 ) .
(d) disc p o l y a c r y l a m i d e gel (0.5 c m x 1 0 c m ) s h o w i n g a m i d o b l a c k
s t a i n i n g p r o t e i n s (Top to b o t t o m , H 3 , H 2 B , H2A and H 4 ) from main
n u c l e o s o m e b a n d ; the m i n o r c o m p o n e n t a l s o c o n t a i n s the same
histones.
s o l u b l e f r a c t i o n s , shown in F i g . 2 B , i n d i c a t e s a s ionic s t r e n g t h
i n c r e a s e s t h e r e is a p r e f e r e n t i a l s e l e c t i o n o f m o n o n u c l e o s o m e s
o v e r o l i g o m e r i c f o r m s and a r e d u c t i o n in h e r e r o g e n e i t y of m o n o n u c l e o s o m e s . T o f u r t h e r q u a l i f y the s i g n i f i c a n c e o f the i n d i vidual m o n o n u c l e o s o m e c o m p o n e n t s w i t h r e s p e c t t o p r o t e i n , DNA
size and t h e r m a l m e l t i n g b e h a v i o u r , n u c l e o s o m e s w e r e p r e p a r a t i v e l y e l e c t r o p h o r e s e d and i n d i v i d u a l c o m p o n e n t s e l u t e d and
e x a m i n e d . F i g u r e 3A s h o w s that the n u c l e o p r o t e i n from the
v a r i o u s b a n d s r e t a i n t h e i r r e l a t i v e n o b i l i t y , w h i c h is a p p r o x i m a t e l y r e l a t e d t o the c o m p u t e d DNA b a s e p a i r n u m b e r . The v a r i a t i o n s are c o n s i s t e n t w i t h o t h e r r e p o r t s ( 1 4 , 2 8 , 4 6 , 4 9 ) . T h e
c o n t e n t o f n o n h i s t o n e is very low (<2%).
T h i s has been a l r e a d y
noted ( 1 4 ) . A s shown in F i g u r e 3 B , the m a j o r v a r i a t i o n in p r o tein c o n t e n t c e n t e r s on w h e t h e r HI a n d / o r H 5 a r e p r e s e n t o r n o t ;
the 125 base pair f r a g m e n t may h a v e m o r e H 3 and H 4 a s s o c i a t e d
w i t h it. D i f f e r e n t i a l s o l u b i l i t y o f the m o n o n u c l e o s o m e p o p u l a t i o n is a p p a r e n t l y r e l a t e d t o HI and H 5 (Fig. 2 ) and some
nonhistone proteins (54).
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Fi g u r e 2. E f f e c t of ionic strength on s e l f - a s s o c i a t i o n or a g g r e g a t i o n of n u c l e o s o m e s . A , low g r a v i t y (2000 x g, 20 m i n u t e s )
s e p a r a t i o n of a g g r e g a t e d n u c e l o p r o t e i n as a f u n c t i o n of salt
concentration.
(A) c h r o m a t i n d i g e s t e d with m i c r o c o c c a l n u c l e a s e
(4 x 1 0 " u n i t s / y g D N A ) for 5 m i n u t e s at 2 3 ° ( 0 . 4 % acid s o l u b l e
( 3 8 ) , 8+ 3% i n s o l u b l e c h r o m a t i n in 1 mM EDTA pH 8.0 at 2 0 0 0 x g
20 m i n u t e s ) . C h r o m a t i n d i g e s t e d to 15 - 18% acid s o l u b i l i t y in
low ( 3 8 , A ) and high ( 1 4 , A ) ionic strength b u f f e r .
Nucleosomes
a s s o c i a t e d with (#) and w i t h o u t (O) histones HI and H5 (see Fig.
3, c and d ) .
N u c l e o s o m e s from 11.5 £ m o n o m e r peak from c h r o m a tin d i g e s t e d at low ionic strength (see A ) and f r a c t i o n a t e d by
5 - 2 0 % s u c r o s e g r a d i e n t s in 1 mM EDTA pH 8.0 (•) or 100 mM N a C l 50 mM T r i s - H C l - 1 mM E D T A , pH 8.0 ( D ) . B_. El e c t r o p h o r e t i c
a n a l y s i s of DNA p r e p a r e d from c h r o m a t i n a g g r e g a t e s c o l l e c t e d in
e x p e r i m e n t , & ; a to j r e f e r to p r e c i p i t a t e s in panel A_; h^ is the
150 mM NaCl s o l u b l e m o n o n u c l e o s o m e . P h o t o g r a p h is a r e v e r s e
p r i n t of a 3 . 5 % p o l y a c r y l a m i d e slab g e l .
Melti ng p r o f i 1 e s : The d e r i v a t i v e melting p r o f i l e s of m o n o n u c l e o somes treated or u n t r e a t e d with urea and/or t r y p s i n are shown in
F i g u r e 4. T h e s e data show that the melting p r o f i l e of isolated
m o n o n u c l e o s o m e s is c o m p o s e d of two c o m p o n e n t s , r e f e r r e d to here
as T" and T 1 " . This a g r e e s with recent r e p o r t s ( 2 2 , 2 3 , 2 7 - 3 5 ) .
Urea t r e a t m e n t , w h i c h has been shown to d i s r u p t c h r o m a t i n and
n u c l e o s o m e s ( 1 6 , 2 3 , 2 7 ) , p r e f e r e n t i a l l y e l i m i n a t e s T" 1 o v e r T " ;
a b o v e 6M urea a D N A - l i k e c o m p o n e n t (T°) o c c u r s (Fig. 4 ) , w h i c h
r e p r e s e n t s a b o u t 8 to 1 2 % of the total h y p e r c h r o m i c i t y .
In our
e x p e r i m e n t s urea n e i t h e r caused a change in total h y p e r c h r o m i c i t y
of the c o m p o n e n t s nor any n o t i c e a b l e increase in t u r b i d i t y ( 2 7 ) .
T r e a t m e n t of m o n o n u c l e o s o m e s w i t h trypsin r e s u l t e d in the
p r e f e r e n t i a l e l i m i n a t i o n of T"' over T". This is also shown in
F i g u r e 4. The e x t e n t of e l i m i n a t i o n of T" could be e n h a n c e d if
urea w a s i n c l u d e d ; urea added b e f o r e or d u r i n g p r o t e o l y t i c d i g e s tion w a s very e f f e c t i v e in this e l i m i n a t i o n . T r y p s i n and urea
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b
265
2F5
c
d
210
165
165
e
f
145
125
Figure 3. S u b f r a c t i o n a t i o n and protein c h a r a c t e r i z a t i o n o f mononucleosomes. A., p r e p a r a t i v e electrophoresis o f a low speed
fraction of micrococcal nuclease digested chromatin (0.05 ionic
strength soluble fraction in Fig. 2 (A) and ref. 1 4 , Fig. l a j .
The 3.5% (1 cm x 2 0 c m ) polyacrylamide slab gel contained 2.5 to
3.0 mg of sample per 3 cm slot. Electrophoresis was for 1 4 hours
at 3 0 mA. Ethidium bromide fluorescent bands were exercised and
contents e l e c t r o p h o r e t i c a l l y eluted. The lower slab (0.3 cm x
20 cm) shows the purity o f the components. The base pair a s s i g n ments of the DNA from each component were from calibrated fragments (Fig. l c ) . J3, densitometric scans (575 n m ) of histones
from each component. Electrophoresis was at pH 2.7 ( 4 3 , 4 4 ) .
treatment generated two main types o f DNA m e l t i n g : one with
melting properties e q u i v a l e n t to deproteinized DNA and a second,
broad melting component o f value <T". The latter can only be
completely removed by addition of sodium d o d e c y l s u l p h a t e and
further DNA purification (Fig. 4 ) . In all e x p e r i m e n t s , using
the same nucleosomal starting material and a p p r o p r i a t e blank
c o n t r o l s , the difference in total hyperchromicity between n a t i v e ,
urea, and urea-trypsin treatments was less than 6.8%. Trypsin
treatment alone was about 8.0%. The sharp c o - o p e r a t i v e character
and near equivalent hyperchromic values suggest that no major
single strand stacking has taken p l a c e , except possibly for
trypsin treated m a t e r i a l .
Figure 5 shows the results of a study using the e l e c t r o phoretically fractionated mononucleosomes of Figure 3 and o l i g o mers from the ionic strength precipitation experiments (Fig. 2 ) .
Firstly, it is apparent from the thermal d e r i v a t i v e plots that
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10.0
I 0
30 50 70 90
Temperature °C
Fi gure 4. D e r i v a t i v e thermal d e n a t u r a t i o n p r o f i l e s of m o n o n u c l e o somes before and after treatment with urea a n d / o r t r y p s i n . T h e
p l o t s (24) w e r e o b t a i n e d with 1.5 x 10' 1 * M DNA-P in 5 mM P d , a t
pH 7.0. Mononucl e o s o m e s a r e from e x p e r i m e n t in Fig. 1. a_, u n t r e a t e d ; b_, urea 6 M f o r two hours and c_, 8 M ; d_, trypsin 0.003
w/w at 2 3 ° for 1 hour at pH 7.0 and e_ trypsin with 6 M urea
added during d i g e s t i o n , and £ , D N A from n u c l e o s o m e s in 6 M u r e a .
E x p e r i m e n t s w e r e in d u p l i c a t e ; d e t a i l s a r e in M e t h o d s .
m e l t i n g of n u c l e o s o m e fragments are s i m i l a r . S e c o n d l y , as DNA
f r a g m e n t size is i n c r e a s e d there is a slight shift of the T'"
t r a n s i t i o n and an a p p e a r a n c e of minor low (<T") and high ( > T M I )
m e l t i n g t r a n s i t i o n s . T h i r d l y , c o m p o n e n t T" is slightly augmented
and less c o - o p e r a ; i v e (measured by half peak h e i g h t ) in submonomer p a r t i c l e s (the; 1 2 5 base pair u n i t ) ; it b e c o m e s increasingly
c o n t i g u o u s with T"' as D N A fragment size i n c r e a s e s . Since some
f r a g m e n t s have e i t h e r HI or H5 a s s o c i a t e d with t h e m , the shift of
T" and T" 1 may be d u e to these p r o t e i n s . H o w e v e r , T" broadens and
both T" and T"' s h i f t to lower values when 1 mM P 0 4 buffer is used
C o n t r o l s for c o r r e l a t i n g visual and thermal s t u d i e s : As major
c o n t r o l s w e p r e v i o u s l y studied t h e patterns and images found in
the a b s e n c e o f s a m p l e , stability of s a m p l e to uranyl salt conc e n t r a t i o n and c o n t a m i n a t i o n due to free histone ( 1 4 ) . T h e
d r y i n g pattern w i t h b u f f e r alone p r e p a r e d and stained in the
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10.0 -
30 50 70
90
Temperature °C
F i g u r e 5. D e r i v a t i v e thermal d e n a t u r a t i o n p r o f i l e s o f m o n o n u c l e o s o m e s , d i n u c l e o s o m e s and o l i g o m e r i c f r a g m e n t s . Plots a_ and b^
c o r r e s p o n d to f r a g m e n t s o b t a i n e d f r o m e x p e r i m e n t s in Fig. 2 T n d i cated by s y m b o l s A and A ; c t o f p l o t s c o r r e s p o n d to b_, c_, d_, a n d
f_ c o m p o n e n t s in Fig. 3.
s a m e w a y as for s a m p l e s , is n e i t h e r r a n d o m nor the same as w h e n
s a m p l e is i n c l u d e d . The d e p o s i t i o n p a t t e r n is a l t e r e d as the
c o n c e n t r a t i o n o f sample per grid is i n c r e a s e d ( 1 4 ) . A t h i g h e r
m a g n i f i c a t i o n s no images c o m p a r a b l e to the ones for n u c l e o s o m e s
c o u l d b e d e t e c t e d w i t h b u f f e r a l o n e a t any t e m p e r a t u r e . T h i s
possibility was extensively studied because transfer of heated
s a m p l e from thermal c u v e t t e to i n d i v i d u a l e l e c t r o n m i c r o s c o p e
grids could i n t r o d u c e a r t i f a c t s due to heat f l u c t u a t i o n s o f
s a m p l e and g r i d . The v a r i a b l e s a s s o c i a t e d w i t h sample t r a n s f e r
w e r e q u a l i f i e d for each i n c r e a s e in t e m p e r a t u r e by use o f m i c r o t h e r m o c o u p l e s i n s e r t e d a p p r o p r i a t e l y in the c a p i l l a r i e s o f the
t e m p e r a t u r e e q u i l i b r a t e d m i c r o p i p e t t e s and o n t o the grid s u r f a c e s .
A detail o f this d e s c r i p t i o n is p r e s e n t e d in the M e t h o d s and in
the legend o f F i g u r e 6. From F i g u r e 6A and C it can be seen
that the a v e r a g e time r e q u i r e d for s a m p l e t r a n s f e r and t r e a t m e n t
on the grid is very s h o r t , 1.63 ± 0.3 s e c o n d s and 2.13 ± 0.2
s e c o n d s , r e s p e c t i v e l y . Loss o f heat d u r i n g t r a n s f e r is c h a r a c 2241
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A
90
-.
eo
—»
70
70
90
50
Temperature Before
Transfer (°C)
60
50
40
30
20
I
2
3
Time (seconds)
I
2
Duration (seconds)
Fi g u r e 6_. D e t e r m i n a t i o n o f t e m p e r a t u r e d i f f e r e n t i a l s d u r i n g
s p e c i m e n h e a t i n g a n d t r a n s f e r t o e l e c t r o n m i c r o s c o p e g r i d s . A_.
C h a n g e in s a m p l e t e m p e r a t u r e d u r i n g t r a n s f e r f r o m t h e r m a l c u v e t t e
to i n d i v i d u a l g r i d s . V e r t i c a l s h a d e d bar i n d i c a t e s all p o i n t s
w h e r e s a m p l e s w e r e r e l e a s e d from 5 m i c r o l i t r e p i p e t t e s e q u i p p e d
w i t h m i c r o - t h e r m o c o u p l e p r o b e s i n the c a p i l l a r i e s . ]3. D i s c r e p e n c y in s a m p l e t e m p e r a t u r e b e f o r e t r a n s f e r and i n c i p i e n t r e l e a s e
o f s a m p l e o n t o e l e c t r o n m i c r o s c o p e g r i d s . Data w a s o b t a i n e d
f r o m g r a p h s in A u s i n g a n a v e r a g e d t r a n s f e r t i m e o f 1.63 +_ 0.30
s e c , w h e r e the n u m b e r o f r e p e t i t i o n s w a s 8 5 . C_. R e s u l t a n t
increased grid t e m p e r a t u r e s following application o f samples a t
v a r i o u s s a m p l e t r a n s f e r t e m p e r a t u r e s s h o w n in A and B_. V e r t i c a l
t e m p e r a t u r e d r o p s i n d i c a t e p o i n t s in t i m e and T i n a l grid t e m p e r a tures when w a s h i n g o f grids with staining solutions o r w a t e r
o c c u r r e d . Maximum grid t e m p e r a t u r e , which occurs at 95°C, w a s
32.4 + 0.8°C.
t e r i s t i c for e a c h s t a r t i n g t e m p e r a t u r e ; it d o e s not b e c o m e s i g n i f i c a n t until a b o v e 7 0 ° C ( F i g . 6 A and B ) . A t 9 7 ° C the a v e r a g e
h e a t l o s s d u r i n g t r a n s f e r w a s 2 . 8 ± 1 . 6 ° C . S i m i l a r l y it w a s
f o u n d t h a t s a m p l e t r a n s f e r t e m p e r a t u r e b e l o w 5 0 ° C did n o t
s i g n i f i c a n t l y a l t e r the o r i g i n a l g r i d t e m p e r a t u r e ; m a x i m u m g r i d
t e m p e r a t u r e at 97°C was 8.4 + 0.8°C above ambient room t e m p e r a t u r e . E v e n a t t h i s t e m p e r a t u r e the e x p o s u r e w a s less t h a n 1.5
s e c o n d s after which the grids w e r e flushed with buffer (Fig. 6 C ) .
By u s i n g the d a t a in F i g u r e 6 a n d j u d i c i o u s l y c o m p a r i n g g r i d s
w i t h o u t sample, before and after h e a t i n g , we concluded that
elevated temperatures alone cannot generate morphological staining a r t i f a c t s .
C o o l i n g o f s a m p l e d u r i n g t r a n s f e r could r e s u l t in p o s s i b l e
r e n a t u r a t i o n o f s o m e d e n a t u r e d m a t e r i a l . To d e t e r m i n e to w h a t
e x t e n t this m i g h t o c c u r c h r o m i c i t y c h a n g e s a t 2 6 0 nm w e r e m o n i tored a s n u c l e o s o m e samples were program heated to various
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100 -
80
= 60
S
I
40
X
20
-
if"
f1 -_
J
^
|
j
40
60
80
Temperature CO
Figure 7. A n a l y s i s o f n u c l e o s o m a l - D N A d e n a t u r a t i o n a n d r e n a t u r a t i o n . S a m p l e s o f n u c l e o s o m e s (1.5 x 1 0 " 4 M D N A - P ) , in t r i p l i c a t e , w e r e m e l t e d to v a r i o u s e x t e n t s ( a , b a n d c ) , c o o l e d
r a p i d l y to a m b i e n t t e m p e r a t u r e ( 2 3 ° ) a n d r e - h e a t e d by w a y o f a
thermal p r o g r a m to 9 7 ° . H y p e r c h r o m i c i t y o f c o n t r o l s a m p l e , d e natured in o n e c o n t i n u o u s phase w a s 3 3 . 7 % (
) . S y m b o l s (-<—)
and (—>--) i n d i c a t e c o o l i n g a n d r e - h e a t i n g p h a s e s , r e s p e c t i v e l y .
t e m p e r a t u r e s , c o o l e d to room t e m p e r a t u r e a n d p r o g r a m r e - h e a t e d
to 97° ( m e l t i n g o f all D N A ) . T h e r e s u l t s , p r e s e n t e d in Figure 7 ,
are only f o r t h e b e g i n n i n g s a n d ends of t h e T " a n d T"' t r a n s i t i o n s . Each o f t h e t r a n s i t i o n s h a s some p o t e n t i a l f o r h y p o c h r o m i c i t y (after c o r r e c t i n g f o r w a t e r e x p a n s i o n ) ; the b e g i n n i n g
of t h e T " t r a n s i t i o n is m o s t a f f e c t e d a n d T"' t h e l e a s t .
H y p e r c h r o m i c i t y d u e to r e - h e a t i n g the n u c l e o s o m a l m a t e r i a l is
almost totally recoverable for the former, b u t not for the
l a t t e r . T h e r e f o r e c o m b i n i n g t h e r e s u l t s in F i g u r e 6 A , B a n d C
with those o f F i g u r e 7 , it is likely that only t h e m a t e r i a l at
the b e g i n n i n g o f t r a n s i t i o n T " m i g h t be a f f e c t e d by t r a n s f e r o f
s a m p l e d u r i n g h e a t i n g . H o w e v e r , a t these t e m p e r a t u r e s s a m p l e
heat l o s s , p r i o r to f i x a t i o n on t h e g r i d s , is very s m a l l .
To d e t e r m i n e to w h a t e x t e n t the m o r p h o l o g i c a l a n d s p e c t r o scopic c h a n g e s , which occur during nucleosomal-DNA d e n a t u r a t i o n ,
may be a t t r i b u t a b l e to free h i s t o n e - h i s t o n e i n t e r a c t i o n s a l o n e ,
p u r e , n u c l e i c acid f r e e , h i s t o n e s were h e a t t r e a t e d and s i m u l taneously monitored for morphology. The results of these
e x p e r i m e n t s c a n b e b r i e f l y s u m m a r i z e d as f o l l o w s : 1 ) a c o operative heat induced hyperchromic change can be clearly
d e t e c t e d a t 2 7 5 n m ; 2 ) t h e m a j o r t r a n s i t i o n o c c u r s a b o u t 10°C
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Table 1.
Form
S t a t i s t i c a l measurements of native and denatured Mononucleosomes
N
O.D.
I.D.
(nm + s.d.)
Native
550
13.3 + 1.5
Denatured
4 0 5 17.4 + 2.2
Contour Length a
(nm + s.d.)
Percent B-Form
Length
1.68+0.8
9.8 + 2.0
42.9 + 6.3
86 + 4.8
C o m p u t e d b y d i r e c t l e n g t h m e a s u r e m e n t o f l i n e a r f o r m s o r by
f o r m u l a , T T ( ( O . D . - I.D.)/2 + I . D . ) .
C o m p u t e d f r o m w e i g h t a v e r a g e m o l e c u l a r w e i g h t 9 8 , 3 4 0 d +_ 3 % .
a b o v e t h a t o f p u r i f i e d DNA from n u c l e o s o m e s ; a n d 3) the m o r p h o l o g y
o f t h e h i s t o n e s , w h i c h h a v e been d e s c r i b e d ( 1 4 ) , c h a n g e , but the
i m a g e s d o n o t r e s e m b l e t h o s e o b t a i n e d w i t h n u c l e o s o m e s a t any o f
the elevated t e m p e r a t u r e s .
Image and s t a i n i n g c h a r a c t e r i s t i c s o f native and d e n a t u r e d nucleosomes : Figure 8 shows the general n e g a t i v e s t a i n i n g properties
of m o n o n u c l e o s o m e s at 2 3 ° C , prior to thermal d e n a t u r a t i o n . All
images were p a r t i c u l a t e , either circular o r prolate e l i p s o i d s .
A s i z e a n a l y s i s o f t h e i n s i d e and o u t s i d e d i a m e t e r s i s s u m m a r i z e d
in T a b l e I. T h e d i m e n s i o n s of t h e p a r t i c l e s a r e i n g o o d a g r e e m e n t
w i t h o u r e a r l i e r d e t a i l e d study ( 1 4 ) and t h o s e o b t a i n e d from c h i c k
e r y t h r o c y t e s (1,2,11). T h e bulk o f t h e n u c l e o s o m a l i m a g e s c h a n g e d
v e r y l i t t l e i n s h a p e o r s t a i n a b i l i t y a s t h e t e m p e r a t u r e is r a i s e d
f r o m a m b i e n t ( 2 3 ° C ) t o 7 0 ° C . T h i s h o l d s w h e t h e r t h e s a m p l e is
fixed in g l u t e r a l d e h y d e immediately prior to s t a i n i n g o r directly
s t a i n e d w i t h o u t f i x a t i o n (Fig. 8 b , b ' ) . A s i g n i f i c a n t a m o u n t o f
s w e l l i n g and tendency of the nucleosomes to aggregate was noticed
at t h e l a t t e r p a r t o f t r a n s i t i o n T" ( F i g . 8 b , b ' ) . H o w e v e r , no
f u r t h e r c h a n g e w a s n o t e d until T " ' w a s r e a c h e d ; a t t h i s p o i n t ,
t h e a v e r a g e n u c l e o s o m a l m o r p h o l o g y has d r a s t i c a l l y a l t e r e d and
both inner and outer diameters have increased (Fig. 8c,c',
Table I ) , The average fiber length from p e r i m e t e r measurements
is l e s s t h a n t h e l e n g t h c o m p u t e d f r o m t h e w e i g h t a v e r a g e d b a s e
pair number a s s u m i n g B-form duplex DNA (Fig. 1 ) ( 1 , 2 7 ) .
A d e t a i l e d c o m p a r i s o n of the u n f i x e d a n d f i x e d m a t e r i a l
i n d i c a t e s t h a t t h e u n f i x e d m a t e r i a l y i e l d s m o r e l i n e a r and U 2244
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a
Figure 8. Morphological changes in mononucleosomes at various
stages of heat denaturation. Duplicate samples removed from
thermal-regulated cuvette at 2°C intervals and transferred to
grids. Procedures are described in methods and in Fig. 6. a_,
a}, 2 3 ° ; b^ b} and c_, c_l correspond to end of T" and T"' transitions shown in Fig. 4., d, d' are high magnifications of c,
c'. Gl uteral dehyde was omitted in a', b', c 1 and d_'.
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s h a p e d s t r u c t u r e s than o p e n ended c i r c l e s . This is s h o w n in
the high m a g n i f i c a t i o n s o f F i g u r e 8 d , d ' . Close s c r u t i n i z a t i o n
of g r i d s with s a m p l e s from each t e m p e r a t u r e interval i n d i c a t e d
that a m i n o r c o m p o n e n t w i t h similar m o r p h o l o g i c a l f e a t u r e s seen
in F i g u r e 8 b to c also o c c u r s a t the b e g i n n i n g o f T". H o w e v e r ,
t h e s e ringed s t r u c t u r e s may have d i s i n t e g r a t e d as the t e m p e r a t u r e
w a s i n c r e a s e d , b e c a u s e they could not be detected a t h i g h e r temperatures.
DISCUSSION
The c o m p o s i t i o n o f m o n o n u c l e o s o m e s used here s p e c i f i c a l l y
for c o m b i n e d m e l t i n g and v i s u a l i z a t i o n studies is in good a g r e e m e n t w i t h o t h e r r e p o r t s ( 3 - 6 ) . We have shown that m o n o n u c l e o some v a r i a t i o n ( 1 4 , 2 7 ) can arise through d i f f e r e n c e s in s e l f a s s o c i a t i o n o f m o n o m e r o r o l i g o m e r n u c l e o s o m e s by i n c r e a s e d ionic
s t r e n g t h and p r e s e n c e o f HI and H5 a s s o c i a t e d p r o t e i n s . T h e
h e t e r o g e n e i t y e s t a b l i s h e d by e l e c t r o p h o r e t i c a l l y s u b f r a c t i o n a t e d
m o n o n u c l e o s o m e s is in a g r e e m e n t with those from c h i c k e n ( 2 7 ) .
The e x t r e m e l y low n o n h i s t o n e p r o t e i n c o n t e n t o f the m o n o m e r s is
not unusual b e c a u s e : 1) m a t u r e c h i c k e n or goose e r y t h r o c y t e
nuclei c o n t a i n very low a m o u n t s o f n o n h i s t o n e in c o m p a r i s o n to
nuclei from i m m a t u r e c e l l s or o t h e r tissues ( 5 2 , 5 3 ) ( e s p e c i a l l y
in c l a s s e s b e l o w 6 8 , 0 0 0 d a l t o n s ) and 2) the bulk o f the high
m o l e c u l a r w e i g h t r e s i d u a l p r o t e i n s , which a p p e a r to be m o s t l y
a s s o c i a t e d w i t h n u c l e a r m e m b r a n e s o r m a t r i x , are r e l a t i v e l y
i n s o l u b l e u n l e s s e x t e n s i v e l y d i g e s t e d with m i c r o c o c c a l n u c l e a s e
( 5 4 ) . Since the initial p r e p a r a t i o n o f this m a n u s c r i p t several
r e l a t e d reports have been p u b l i s h e d which f u r t h e r d o c u m e n t the
d i f f e r e n t i a l s e l f - a s s o c i a t i o n o f m o n o m e r s and o l i g o m e r s ( 3 2 , 5 5 - 3 7 )
T h e s e s t u d i e s p r o v i d e a basis for i n t e r p r e t i n g the e a r l i e r
observed differential sensitivity o f erythrocyte chromatin
( h e t e r o c h r o m a t i c and e u c h r o m a t i c s t r u c t u r e ) to both m o n o and
d i v a l e n t c a t i o n s ( 5 7 - 6 0 ) b e f o r e and a f t e r s e q u e n t i a l removal o f
p r o t e i n s (.52,53,59).
The m e l t i n g p r o f i l e s o f e l e c t r o p h o r e t i c a l l y d e f i n e d m o n o n u c l e o s o m e s and s e l e c t o l i g o m e r i c f r a g m e n t s o b t a i n e d by i o n i c
s t r e n g t h p r e c i p i t a t i o n m e t h o d s c l e a r l y i n d i c a t e that o n l y two
m a j o r t r a n s i t i o n s (T" and T"') are a s s o c i a t e d with m o n o n u c l e o s o m e s . As s h o w n here and in p a r t e l s e w h e r e ( 2 2 , 2 3 , 2 7 , 2 8 , 3 0 - 3 4 ) ,
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t h e r m a l t r a n s i t i o n s £ T " a n d > T " ' a r e p e c u l i a r to n u c l e o p r o t e i n s of
l a r g e D N A l e n g t h (>_ 2 6 5 b a s e p a i r s ) a n d i n c r e a s e d p r o t e i n c o m p l e x i t y .
H o w e v e r they a r e n o t n e c e s s a r i l y d u e to p r e v i o u s d e g r a d a t i o n ( 2 1 ,
2 5 , 2 8 , 3 0 , 3 2 ) . Allowing for solvent d i f f e r e n c e s , o u r results a r e
very s i m i l a r t o t h o s e o b t a i n e d by M a n d e l a n d F a s m a n ( 2 2 ) , L e w i s
( 2 8 ) , W i t t i g a n d W i t t i g ( 3 2 ) . T h e y a r e a l s o in e x c e l l e n t a g r e e m e n t w i t h t h e h i g h l y c o m p l e m e n t a r y p h y s i c a l s t u d i e s by W e i c h e t a n d
co-workers (68) that appeared on submission date o f our original
manuscript. The combined studies clearly indicate that the presence
of t r a n s i t i o n s T " a n d T " ' c a n n o t b e d i r e c t l y a c c o u n t e d f o r by
v a r i a t i o n s in D N A l e n g t h , p r e s e n c e o r a b s e n c e o f l y s i n e - r i c h
histones (HI, H5) or nonhistone proteins; but, co-operativity and
r e s o l u t i o n o f t h e s e t r a n s i t i o n s p a r t i c u l a r l y T " , c a n be i n f l u e n c e d
by t h e p r e c e d i n g p a r a m e t e r s a s well as by d i f f e r e n c e s in i o n i c
s t r e n g t h o f the m e l t i n g b u f f e r . W e r o u t i n e l y u s e d 5 mM N a P O . a t
pH 7 . 0 , b e c a u s e m u c h less v i s u a l u n f o l d i n g o f t h e m o n o n u c l e o s o m e s
( 1 4 ) t a k e s p l a c e t h a n a t l o w e r i o n i c s t r e n g t h s s u c h a s w i t h 0.1 m M
E D T A , a n d T " a n d T " ' a r e s t i l l d i s t i n c t . W e i c h e t e t a l ( 6 8 ) have
also recently observed t h e latter effect using sodium cacodylate
buffer. T h e thermal reversibility o f T" and differential sensitivity of T"' with urea and trypsin treatment o f n u c l e o s o m e s will
be d i s c u s s e d f u r t h e r in r e f e r e n c e to n u c l e o s o m e u n f o l d i n g .
Olins a n d c o - w o r k e r s (27) have skillfully shown by electron
microscopy that urea induces unfolding of n u c l e o s o m e s . A s i m i l a r
c l a i m has b e e n m a d e by W o o d c o c k a n d F r a d o ( 3 6 ) . S i m p l e s t o r a g e o f
n u c l e o s o m e s in l o w i o n i c s t r e n g t h b u f f e r can a l s o p r o m o t e d e n a t u r a tion ( 1 4 , 6 6 ) ; t h e n u c l e o s o m e s u n f o l d into two m a j o r h a l v e s in t h e
a b s e n c e o f any m a j o r D N A o r p r o t e i n d e g r a d a t i o n .
In t h i s s t u d y w e
s h o w for t h e f i r s t t i m e t h a t d u r i n g t h e r m a l d e n a t u r a t i o n p u r i f i e d
mononucleosomes undergo reproducible morphological changes which
are g e n e r a l l y n o n r e v e r s i b l e . F i r s t , the m o r p h o l o g i c a l c h a n g e s o c c u r
w h e t h e r o r n o t t h e n u c l e o s o m e s a r e f i x e d in g l u t e r a l d e h y d e p r i o r to
u r a n y l a c e t a t e s t a i n i n g . S e c o n d , t h e m a j o r c h a n g e is f r o m t h e
typical compact toroid shape (Fig. 8 , Table I and ref. 2 , 7 , 1 0 ,
1 1 - 1 5 ) to t h e l a r g e c i r c u l a r , U - s h a p e d , c r e s c e n t - s h a p e d , o r e x t e n d e d
structure o f length about 9 0 % o f a weight average native D N A
B-form equivalent. T h i r d , the major portion o f these morphological
c h a n g e s is seen o n l y a f t e r r e a c h i n g t r a n s i t i o n T " ' . U n l i k e f o r m a i -
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d e h y d e (.67), w h i c h w a s p u r p o s e l y a v o i d e d h e r e , w e do n o t feel t h e
highly regular ring structures obtained with gluteraldehyde fixation
a r e a r t i f a c t s ; b a s i c a l l y t h e s a m e i m a g e s w e r e o b t a i n e d by t w o
independent methods of staining. H o w e v e r , the presence of l i n e a r ,
e x t e n d e d f o r m s in u n f i x e d s a m p l e s s u p p o r t s o u r i n i t i a l c o n t e n t i o n
that fixation would help stabilize the delicate denatured nucleosome structures from mechanical disruption during staining, washing
and drying of g r i d s . The inherent r i g i d i t y of small DNA segments
o f t h i s o r d e r ( 5 0 ) , p a r t i c u l a r l y in t h e d e n a t u r e d s t a t e , w o u l d t e n d
to g e n e r a t e l i n e a r s t r u c t u r e s s u c h a s a l r e a d y s e e n f o r l o w i o n i c
s t r e n g t h (14,66) and urea (27,36) d e n a t u r e d n u c l e o s o m e s . A d e t a i l e d
s t u d y o f t h e u n f o l d e d s t r u c t u r e s a t h i g h r e s o l u t i o n , a n d an
a n a l y s i s of the p e r i o d i c , n e g a t i v e l y s t a i n i n g e l e m e n t s , which are
s e e n a l o n g t h e o p e n r i n g e d i m a g e s , is b e i n g c a r r i e d o u t u s i n g i m a g e
r e c o n s t r u c t i o n m e t h o d s . The ringed structures are highly compatible
with recent X-ray diffraction data (15) and corresponding subunit
models (37,64).
Several studies (21,22,26-30,61,68, this study) indicate
t h a t t h e m a j o r m e l t i n g c o m p o n e n t of c h r o m a t i n is l i k e l y r e l a t e d
to t h e p r i n c i p a l t r a n s i t i o n ( T " ' ) o f t h e s i m p l e m o n o n u c l e o s o m e .
H o w e v e r , t h e s h e a r c o m p l e x i t y o f D N A - h e l i x s t a b i l i t y in c h r o m a t i n
p r o h i b i t s d e f i n i t i v e a s s i g n m e n t o f o t h e r m e l t i n g t r a n s i t i o n s at
t h i s t i m e . N e v e r t h e l e s s , an e a r l i e r m o d e l , d e r i v e d f r o m c h r o m a t i n
w o r k ( 1 8 ) , h a s c o n s i d e r a b l e a p p e a l in t h a t t h e t w o m a j o r t r a n s i t i o n s a r e p o s t u l a t e d to o r i g i n a t e f r o m t h e d i s r u p t i o n o f p r o t e i n p r o t e i n i n t e r a c t i o n s a n d m e l t i n g of p r o t e i n - f r e e D N A (Tm ) a n d f r o m
t h e d e s t r u c t i o n o f t h e s u p e r c o i l and D N A s t r a n d s e p a r a t i o n (Tm ) .
O u r visual and h y p e r c h r o m i c studies and the r e c e n t l y r e p o r t e d
h y p e r c h r o m i c , c i r c u l a r d i c h r o i c and c a l o r i m e t r i c s t u d i e s on c h i c k e n
e r y t h r o c y t e n u c l e o s o m e s by W e i c h e t £jt aj_ ( 6 8 ) p r o v i d e n e w i n s i g h t
into the thermal denaturation process of chromatin subunits.
O v e r a l l , t h e b i - p h a s i c m e l t i n g o f c o r e p a r t i c l e s is i n t e r p r e t e d
a s a d e n a t u r a t i o n o f a b o u t 4 0 b a s e p a i r s in t h e f i r s t p h a s e ,
f o l l o w e d by a m a s s i v e u n f o l d i n g o f t h e . n a t i v e s t r u c t u r e of a
t i g h t h i s t o n e - D N A c o m p l e x , which frees the remaining 100 base
p a i r s in s t a c k i n g ( t h e s e c o n d p h a s e ) . O u r s t u d y c l e a r l y s h o w s
t h a t n u c l e o s o m e s b e g i n to alter original t o r o i d s h a p e only d u r i n g
t h e l a t t e r t h i r d o f T " a n d do n o t a c t u a l l y c o - o p e r a t i v e l y u n f o l d
u n t i l T " 1 is r e a c h e d . T h e u n f o l d i n g p a t t e r n is h i g h l y c o n s i s t e n t
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with the predicted 1 3/4 loop or supercoil arrangement of nucleosomal DNA ( 1 5 , 3 7 ) . From the circular dichroic data (68) it is
further apparent that no change in secondary structure of the
protein core takes place at T". Since the core is s t a b l e , it
therefore may serve to lock the major part of the DNA into a rigid
complex thus allowing only certain regions to gain limited mobility
at £ T". This would explain the reversibility of T" that we both
o b s e r v e , albeit in our case complete hypochromicity occurred only
in the first third of T". It is important to stress that the first
stable morphologically altered state of the nucleosome occurs in
the latter third of T". It is there that the toroids become
measureably expanded, frequently aggregated into clusters of 2 to
4 and ends of loops are occasionally detectable. Since secondary
structure of histones is located in the more hydrophobic d o m a i n s ,
which are the regions that interact with each other ( 3 - 6 , 2 8 , 3 1 , 6 1 ,
6 3 , 6 5 , 6 8 ) , the aggregation property may be a direct measurement of
the alteration in histone structure that takes place at this
stage C 6 8 ) .
Unfolding of nucleosome secondary structure by urea has
been earlier shown by electron microscopy ( 2 7 , 3 5 , 6 0 ) , hydrodynamic
and circular dichroic studies ( 2 3 , 3 5 , 6 0 ) . Physical changes in
mononucleosomes following trypsin treatment have also recently been
reported ( 3 1 ) . The data are explained on the basis of a structural
unfolding of nucleosomal DNA whereby part of the DNA rcetains the
properties within intact core particles and the remaining portion
behaves much like free DNA. We note that the melting data of 01 ins
and co-workers (27) and Lilley and Tachell (31) exhibit a similar
preferential loss of transition T"' over T" that we observed here.
However, no visual studies on the unfolding of trypsin treated
nucleosomes is currently available for more rigorous interpretation. From present w o r k , which correlates unfolding of the supercoiled DNA with T " 1 , preferential loss of T"' because of u r e a ,
trypsin or urea and trypsin treatment of nucleosomes would be
expected. It is tempting to conclude that the presence of T" 1 can
be used as a direct i n d i c a t o r , at all t i m e s , for m o r p h o l o g i c a l l y
intact nucleosomes. H o w e v e r , our recent shearing studies (in
preparation) indicate that this is not always t r u e , particularly
if no T" is present. Similar caution should be applied when inter-
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p r e t i n g the o r i g i n of t r a n s i t i o n s <_ T". In a d d i t i o n to T" a r i s i n g
f r o m t h e m e l t i n g of a l i m i t e d n u m b e r of b a s e pairs w i t h i n the
n u c l e o s o m e ( 6 8 ) , it c o u l d also a r i s e from the m e l t - o u t of a m i n o r
n u c l e o s o m e or n u c l e o s o m e - 1 i k e p o p u l a t i o n w h i c h lacks thermal
s t a b i l i t y in the d e n a t u r e d state as e x h i b i t e d by d e n a t u r e d core
n u c l e o s o m e s a t t e m p e r a t u r e s > T " ' . C l o s e s c r u t i n i z a t i o n of
m i c r o g r a p h s o b t a i n e d from the e a r l y p o r t i o n of T" of a n u m b e r of
s a m p l e s i n d i c a t e s that this is p o s s i b l e . T h e r e f o r e , for some
n u c l e o s o m e e l e m e n t s t h e r e may be a d i f f e r e n c e in e i t h e r the types
of p r o t e i n ( s p e c i e s or c h a r g e m o d i f i c a t i o n s ) or t h e way core
p r o t e i n s a r e a s s o c i a t e d w i t h DNA s i s t e r s t r a n d s (intra or i n t e r s t r a n d c r o s s - 1 i n k i n g ) a f t e r d e n a t u r a t i o n . A study of s i n g l e
s t r a n d a v a i l a b i l i t y and DNA s e q u e n c e p o l a r i t y b e f o r e , d u r i n g and
a f t e r thermal d e n a t u r a t i o n of n a t i v e n u c l e o s o m e s may be useful
a p p r o a c h e s for i n v e s t i g a t i n g this p o s s i b i l i t y .
ACKNOWLEDGEMENTS
The a u t h o r s w i s h to thank D r s . C.V. L u s e n a ( N . R . C . C . ) and
A . R . M o r g a n ( U n i v e r s i t y of A l b e r t a ) for p r o v i d i n g us with X and
P M 2 D N A . We a r e e x t r e m e l y i n d e b t e d to the e x p e r t technical
s u p p o r t s k i l l s of M . J . D o v e , L. S o w d e n and R. W h i t e h e a d .
This
is N R C C c o n t r i b u t i o n N o . 16572.
REFERENCES
1 O l i n s , D . E . and O l i n s , A . L . ( 1 9 7 4 ) S c i e n c e 1 8 4 , 3 3 0 - 3 3 2
2 O u d e t , P., G r o s s - B e l l a n d , M . and C h a m b o n , P. ( 1 9 7 5 ) Cell 4 ,
281-299
3 K o r n b e r g , R . D . ( 1 9 7 7 ) Ann. Rev. B i o c h e m i s t r y 4 6 , 9 3 1 - 9 5 4
4 F e l s e n f e l d , G. ( 1 9 7 8 ) N a t u r e 2 7 1 , 1 1 5 - 1 2 2
5 L i , H.J. ( 1 9 7 5 ) N u c l e i c Acids R e s . 2 , 1 2 7 5 - 1 2 8 9
6 van H o l d e , K . E . and I s e n b e r g , I. ( 1 9 7 5 ) A c e . Chem. R e s . 8 ,
327
7 van H o l d e , K . E . , S a h a s r a b u d d h e , C.G. and S h a w , B.R.,
van B r u g g e n , E . F . J . and A r n b e r g , A . C . ( 1 9 7 4 ) B i o c h e m . B i o p h y s .
Res. Commun. 6 0 , 1365-1370
8 O l i n s , A . L . , C a r l s o n , D. and O l i n s , D.E. ( 1 9 7 5 ) J. Cell B i o l .
6 4 , 528-537
9 B a k a y e v , V . V . , M e l n i c k o v , A . A . , O s i c k a , V . D . and V a r s h a v s k y ,
A . J . ( 1 9 7 5 ) N u c l e i c Acids R e s . 2 , 1 4 0 1 - 1 4 1 9
10 F i n c h , J . T . , N o l l , M. and K o r n b e r g , R.D. ( 1 9 7 5 ) P r o c . N a t .
Acad. Sci. U.S.A. 7 2 , 3320-3322
11 W o o d c o c k , C . F . L . , S a f e r , J . P . and S t a n c h f i e l d , J.E. ( 1 9 7 6 )
Expt'l Cell R e s . 9 7 , 1 0 1 - 1 1 0
12 L a n g m o r e , J . P . and W o o l e y , J.C. ( 1 9 7 5 ) P r o c . N a t . A c a d . S c i .
U.S.A. 7 2 , 2691-2695
13 P o o n , N . H . and S e l i g y , V . L . ( 1 9 7 7 ) P r o c . of M i c r o s c o p i c a l
S o c i e t y of C a n a d a 4 , 56-57
2250
Nucleic Acids Research
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
P o o n , N.H. and S e l i g y , V . L . (1978) Expt'l Cell Res. 113, 95-110.
F i n c h , J.T., L u t t e r , L.C., R h o d e s , D., B r o w n , R . S . , R u s h t o n ,
B., L e v i t t , M. and K l u g , A. ( 1 9 7 7 ) N a t u r e 2 6 9 , 29-36
A n s e v i n , A . T . , H n i l i c a , L . S . , S p e l s b e r g , T . C . and Kelm, S.L.
(1971) Biochemistry 10, 4793-4803
L i , H.J., C h a n g , C. and W e i s k o p f , M. ( 1 9 7 3 ) B i o c h e m i s t r y 1 2 ,
1763-1772
W i l h e l m , F.X., De M u r c i a , G.M., C h a m p a g n e , M . H . and D a u n e ,
M . P . (1974) E u r . J. B i o c h e m . 4 5 , 4 3 1 - 4 4 3
S a h a s r a b u d d h e , C.G. and van H o l d e , K . E . (1974) J. B i o l . Chem.
249, 152-156
W o o d c o c k , C . F . L . and F r a d o , L.L.Y. ( 1 9 7 5 ) B i o c h e m . B i o p h y s .
Res. Commun. 6 6 , 403-410
M i l l e r , P., K e n d e l l , F. and N i c o l i n i , C. ( 1 9 7 6 ) Nucleic Acids
R e s . 3, 1875-1881
M a n d e l , R. and F a s m a n , G. (1976) N u c l e i c Acids R e s . 3, 1 8 3 9 1855
W h i t l o c k , J.P. and S i m p s o n , R.T. ( 1 9 7 6 ) N u c l e i c Acids
R e s e a r c h 3, 2 2 5 5 - 2 2 6 6
L u r q u i n , P.F. and S e l i g y , V . L . (1976) C h e m . - B i o l . Intera c t i o n s 1 3 , 27-45
L a w r e n c e , J.J., C h a n , D.C.F. and P i e t t e , L.H. ( 1 9 7 6 ) N u c l e i c
A c i d s R e s . 3, 2 8 7 9 - 2 8 9 3
S t a y n o v , D.Z. (1976) Nature 2 6 4 , 5 2 2 - 5 2 5
O l i n s , D . E . , B r y a n , P.N., H a r r i n g t o n , R . E . , H i l l , W . E . and
O l i n s , A . L . ( 1 9 7 7 ) Nucleic A c i d s ' R e s . 4 , 1911-1931
L e w i s , P.N. (1977) C a n . J. B i o c h e m . 5 5 , 7 3 6 - 7 4 6
V e n g e r o v , Y.Y. and P o p e n k o v , V . I . ( 1 9 7 7 ) N u c l e i c Acids Res.
4, 3017-3027
D e f e r , N . , K i t z i s , A . , K r u t h , J., B r a h m s , S. and B r a h m s , J.
(1977) J. N u c l e i c A c i d s R e s . 4 , 2 2 9 3 - 2 3 0 6
L i l l e y , D.M.J. and T a t c h e l l , K. ( 1 9 7 7 ) N u c l e i c A c i d s R e s . 4 ,
2039-2055
W i t t i g , B. and W i t t i g , S. (1977) N u c l e i c A c i d s R e s . 4, 3 9 0 7 3917
C o t t e r , R . I . and L i l l e y , D.M.J. ( 1 9 7 7 ) FEBS L e t t e r s 8 2 , 6 3 - 6 8 .
S t e i n , A . , S t e i n - B i n a , M. and S i m p s o n , R.T. (1977) Proc. N a t .
A c a d . S c i . U.S.A. 7 4 , 2 7 8 0 - 2 7 8 4
G a r r e t , R.A. ( 1 9 7 1 ) B i o c h e m i s t r y 1 0 , 2 2 2 7 - 2 2 3 0
W o o d c o c k , C.F.L. and F r a d o , L.-L.Y. ( 1 9 7 6 ) J. Cell B i o l . 7 0 ,
267a ( A b s t . )
P a r d o n , J . F . , W o r c e s t e r , D.L., W o o l e y , J . C . , C o t t e r , R . I . ,
L i l l e y , D.M. and R i c h a r d s , B.M. ( 1 9 7 7 ) N u c l e i c A c i d s Res. 4 ,
3199-3214.
S o l l n e r - W e b b , B. and F e l s e n f e l d , G. ( 1 9 7 5 ) B i o c h e m i s t r y 1 4 ,
2915B i r n b o i m , H . C . , H o l f o r d , R.M. and S e l i g y , V . L . Cold Spring
H a r b o r S y m p . Q u a n t . B i o l . 42 (in p r e s s )
M a r k o v , G.G. and I v a n o v , I.G. ( 1 9 7 4 ) A n a l . B i o c h e m . 5 9 , 555563
D a h l b e r g , A . E . , D i n g m a n , C.W. and P e a c o c k , A . C . (1969) J.
Mol. Biol. 4 1 , 139-147
L a e m m l i , U. (1970) Nature 2 2 7 , 6 8 0 - 6 8 5
P a n y i m , S. and C h a l k l e y , R. (1969) B i o c h e m i s t r y 8, 3972-3979
T o b i n , R . S . and S e l i g y , V . L . ( 1 9 7 5 ) J. B i o l . C h e m . 2 5 0 , 3 5 8 364.
2251
Nucleic Acids Research
45
G o r d o n , C.N. and K l e i n s c h m i d t , A.K. (1968) B i o c h e m . B i o p h y s .
Acta 155, 305-307
46 01 ins, A . L , , C a r t s o n , R.D., W r i g h t , E.B. and O l i n s , D.E.
(19.76) Nucleic Acids R e s . 3, 3271-3291
47 T o d d , R.D. and G a r r a r d , W . T . (1977) J. B i o l . Chem. 2 5 2 , 4 7 2 9 4738
4 8 V a r s h a v s k y , A . J . , B a k a y e v , V . V . , C h u m a c k o v , P.M. and G e o r g i e v ,
G . P . ( 1 9 7 6 ) N u c l e i c A c i d s R e s . 3, 2 1 0 1 - 2 1 1 4
49. B a k a y e v , V . V . , B a k a y e v a , T.G. and V a r s h a v s k y , A . J . ( 1 9 7 7 )
Cell 11 , 6 1 9 - 6 2 9
5 0 K o v a c i c , R.T. and van H o l d e , K.E. (1977) B i o c h e m i s t r y , 1 6 ,
1490-1498
51 L o h r , D., C o r d e n , J., T a t c h e l l , K., K o v a c i c , R.T. and
van H o l d e , K . E . ( 1 9 7 7 ) P r o c . Nat. A c a d . Sci . U.S.A. 7 4 , 7 8 - 8 3 .
52 S h e l t o n , K.R. and N e e l i n , J.M. (1971) B i o c h e m i s t r y 1 0 , 2 3 4 2 2348
53 S e l i g y , V . L . and Miyagi , M. (1974) E u r . J. B i o c h e m . 4 6 , 2 5 9 269
54 S e l i g y , V . L . , P o o n , N . H . , D o v e , M. and T o b i n , R . S . u n p u b l i s h e d .
55 S a n d e r s , M.M. and H s u , J.T. (1977) B i o c h e m i s t r y 1 6 , 1 6 9 0 - 1 6 9 5 .
56 C a m p b e l l , A.M. and C o t t e r , R . I . (1977) N u c l e i c A c i d s R e s . 4,
3877-3886
57 L i , H.J., H u , A . W . , M a c i e w i c z , R.A., C o h e n , P., S a n t e l l a ,
R.M. and C a n g , C. ( 1 9 7 7 ) Nucleic Acids R e s . 4 , 3 8 3 9 - 3 8 5 4
58 S e l i g y , V . L . and M i y a g i , M. (1969) Expt'l Cell R e s . 5 8 , 2 7 - 3 4 .
59 B r a s c h , K., S e t t e r f i e l d , G. and S e l i g y , V . L . (1971) Expt'l
Cel 1 R e s . 6 5 , 6 1 - 7 2
60 O l i n s , D.E. and O l i n s , A.L. (1972) J. Cell B i o l . 5 3 , 715
61 L i , H.J. ( 1 9 7 7 ) "Chromatin S t r u c t u r e - A M o d e l " in T h e
M o l e c u l a r B i o l o g y of the M a m m a l i a n G e n e t i c A p p a r a t u s , T s ' o ,
P., Ed., p p . 3 2 3 - 3 4 3 . E l s e v i e r / N o r t h - H o l 1 and B i o m e d i c a l P r e s s .
62 W o o d c o c k , C . F . L . ( 1 9 7 7 ) Science 1 9 5 , 1 3 5 0 - 1 3 5 2
63 C a m e r i n i - O t e v o , R.D. and F e l s e n f e l d , G. ( 1 9 7 7 ) N u c l e i c Acids
Res . 4 , 1 1 5 9 - 1 1 8 1
64 W e i n t r a u b , H., Worcel , A. and A l b e r t s , B. (1976) Cell 9,
409-417
65 W e i n t r a u b , H. and van L e n t e , F. (1974) P r o c . N a t . A c a d . S c i .
U.S.A. 7 1 , 4 2 4 9 - 4 2 5 3
66 O u d e t , P., S p a d a f o r a , C. and C h a m b o n , P. Cold Spring H a r b o r
S y m p . Q u a n t . B i o l . 42 (in p r e s s )
67 P o l a c o w , I., C a b a s s o , L. and L i , H.J. (1976) B i o c h e m i s t r y 1 5 ,
4560-4565
68 W e i s c h e t , W . , T a t c h e l l , K., Van H o l d e , K. and K l u m p , H. (1978)
N u c l e i c A c i d s R e s . 5, 1 3 9 - 1 6 0 .
2252