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A Thesis Submitted for the Degree of PhD at the University of Warwick
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A t h e s i s s u b m i t t e d f o r the d e g r e e o f d o c t o r o f p h i l o s o p h y .
D a r r i n P. S m ith.
A n i m a l M o l e c u l a r G e n e t i c s G roup,
D e p a r t m e n t of B i o l o g i c a l Sci e n c e s ,
U n i v e r s i t y of W a r w i c k .
THE BRITISH LIBRARY
D O C UM EN T
su p p lyc e n t r eBRITISH THESES
N O T I C E
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THIS THESIS HAS BEEN MICROFILMED EXACTLY AS RECEIVED
T H E B R IT IS H L IB R A R Y
D O C U M E N T SUPPLY CENTRE
Boston Spa, Wetherby
W est Yorkshire, LS23 7BQ
C O N T E N T S .
T a b l e o f c o n t e n t s . ii
T a b l e of f i g u r e s . X V
A c k n o w l e d g e m e n t s . X X
D e c l a r a t i o n . xxi
ii
T a b l e of contents.
Chapter 1: INTRODUCTION.
1.1 The o c t a m e r p a r a d o x . 1
1 . 1 . 1 T he o c t a m e r m o t i f and i m m u n o g l o b u l i n g e n e
e x p r e s s i o n . 3
1 . 1 . 2 T he o c t a m e r m o t i f and h i s t o n e H 2 B gene
e x p r e s s i o n . 8
1 . 1 . 3 T h e o c t a m e r m o t i f a n d s n R N A g e n e e x p r e s s i o n . 13
1 . 1 . 4 T he S V40 e n h a n c e r c o n t a i n s a n o c t a m e r m o t i f . 15
1 . 1 . 5 The o c t a m e r m o t i f a n d the e x p r e s s i o n o f H S V
a l p h a ( i m m e d i a t e early) g e n e s . 17
1 . 1 . 6 The octaroer m o t i f a n d the e x p r e s s i o n of
o t h e r genes. 18
1 . 1 . 7 The o c t a m e r m o t i f a nd a d e n o v i r u s r e p l i c a t i o n . 20
1.2 M o d e l s of t r a n s c r i p t i o n a l a c t i v a t i o n by
0 c t - l / 0 c t - 2 f r o m t r a n s f e c t i o n s t u d i e s in B
a n d n o n - l y m p h o i d cells. 2 1
1 . 2 . 1 O ct-1 and 0 c t - 2 are t r a n s c r i p t i o n a l
a c t i v a t o r s . 22
1 . 2 . 2 T r a n s f e c t i o n o f S V40 e n h a n c e r - c o n t a i n i n g
c o n s t r u c t s i n t o B a nd n o n - l y m p h o i d c e l l s . 24
1 . 2 . 3 V P 1 6 a n d the a c t i v a t i o n o f U s n R N A g enes. 28
1 . 3 S t u d i e s o n c l o n e d O c t - 1 a n d O c t - 2 A / O c t - 2 B . 31
1 .3.1 C l o n i n g o f O c t - 1 a n d 0 c t - 2 A / 0 c t - 2 B . 32
1 . 3 . 2 O c t - 1 a n d 0 c t - 2 A / B h a v e a c o n s e r v e d P O U d o m a i n
w h i c h d i r e c t s D N A b i n d i n g . 3A
1 . 3 . 3 O t h e r f e a t u r e s d e t e r m i n e d b y e x a m i n a t i o n o f
the s e q u e n c e s o f c l o n e d O c t - 1 a n d 0 c t - 2 A / B . 38
1 . 3 . 4 T r a n s c r i p t i o n a l a c t i v a t i o n b y c l o n e d O c t - 1
a n d 0 c t - 2 A . 40
1 . 3 . 5 T h e O c t - 1 P O U d o m a i n i n t e r a c t s w i t h V P 1 6 . 44
1 . 3 . 6 T h e P O U d o m a i n is s u f f i c i e n t to s t i m u l a t e
D N A r e p l i c a t i o n . 46
1.4 O t h e r P O U d o m a i n a n d o c t a m e r - b i n d i n g p r o t e i n s . 47
1 . 4 . 1 H o m e o b o x - c o n t a i n i n g g e n e s f o r m m u l t i - g e n e
f a m i l i e s in D r o s o p h i l a a n d v e r t e b r a t e s . 47
1 . 4 . 2 T h e u n c- 8 6 g e n e o f e l e g a n s . 49 1 . 4 . 3 T h e p i t u i t a r y - s p e c i f i c t r a n s c r i p t i o n
f a ctor, P i t-1. 49
1 . 4 . 3 A f a m i l y of P O U d o m a i n g e n e s in the m a m m a l i a n
b rain. 54
1 . 4 . 5 A f a m i l y o f o c t a m e r - b i n d i n g p r o t e i n s in
m o u s e e m b r y o g e n e s i s . 55
1 . 4 . 6 O t h e r e x a m p l e s . 58
C h a p t e r 2: M A T E R I A L S .
2.1 G e n e r a l r e a g e n t s . 61
2 .2 S t o c k s o l u t i o n s . 62
2 .3 B a c t e r i o l o g i c a l media. 63
2 . A B a c t e r i a , p l a s m i d s a nd p hage.
2 .A.1 G e n o t y p e s o f E^_ coli s t r a i n s . 6A 2 . 4 . 2 P l a s m i d v e c t o r s . 64
2 . 4 . 3 B a c t e r i o p h a g e v ectors. 65
2 . 4 . 4 P l a s m i d a n d B a c t e r i o p h a g e R e c o m b i n a n t s . 65
C h a p t e r 3: ME T H O D S .
3.1 T i s s u e c u l t u r e .
3 .1.1 G r o w i n g l a e v i s Xtc c e lls. 67
3 . 1 . 2 G r o w i n g m o u s e L cells. 67
3 . 1 . 3 B l o c k i n g X ^ l a e v i s Xtc c e l l s at s t a g e s o f the
c e l l cycle. 67
3.2 O b t a i n i n g o o c y t e s , eggs a n d em b r y o s .
3 .2.1 O o c y t e s . 68
3 . 2 . 2 E g g s a nd e m b r y o s . 68
iv
3 . 4 . 1 F r o m X^_ l a e v i s ooc y t e s . 70
3 . 4 . 2 F r o m a d u l t laevis l i v e r . 71
3 . 4 . 3 F r o m m i c r o i n j e c t e d o o c y t e s . 72
3 . 4 . 4 S e l e c t i o n o f p o l y a d e n y l a t e d R N A b y o l i g o d T
c e l l u l o s e c h r o m a t o g r a p h y . 72
3 . 5 G e l s f o r r e s o l v i n g n u c l e i c a c i d s .
3 . 5 . 1 N o n - d e n a t u r i n g a g a r o s e g els. 73
3 . 5 . 2 L o w m e l t i n g p o i n t a g a r o s e g e l s . 73
3 . 5 . 3 F o r m a l d e h y d e - a g a r o s e R N A g e l s . 74
3 . 5 . 4 D e n a t u r i n g p o l y a c r y l a m i d e g e l s . 75
3 . 6 I s o l a t i o n o f g e n o m i c D N A f r o m a d u l t
X e n o p u s b l o o d . 76
3 . 7 S o u t h e r n b l o t t i n g . 77
3 . 8 N o r t h e r n b l o t t i n g . 79
3 . 9 L arge s c a l e p r e p a r a t i o n o f p l a s m i d D N A a n d
p u r i f i c a t i o n b y c a e s i u m c h l o r i d e / e t h i d i u m
b r o m i d e c e n t r i f u g a t i o n . 80
3.11 R N a s e p r o t e c t i o n a s s a y s . 83
3 . 1 2 S u b c l o n i n g t e c h n i q u e s .
3 . 1 2 . 1 R e s t r i c t i o n e n z y m e di g e s t s . 84
3 . 1 2 . 2 P r e p a r a t i o n o f p l a s m i d v e c t o r s f or s u b c l o n i n g . 84
3 . 1 2 . 3 P r e p a r a t i o n o f t a r g e t D N A f o r s u b c l o n i n g . 85
3 . 1 2 . 4 L i g a t i o n s . 85
3 . 1 3 T r a n s f o r m a t i o n o f E. c o l i w i t h p l a s m i d s .
3 . 1 3 . 1 C a C l2 m e d i a t e d t r a n s f o r m a t i o n . 85 3 . 1 3 . 2 H a n a h a n h i g h e f f i c i e n c y t r a n s f o r m a t i o n . 86
3 . 1 4 P l a t i n g E_j_ c oli t r a n s f o r m e d o r i n f e c t e d w i t h
b a c t e r i o p h a g e M 1 3 . 88
3 . 1 5 S m a l l s c a l e i s o l a t i o n of p l a s m i d D N A a nd M 1 3
R F DNA. 88
3 . 1 6 P r e p a r a t i o n o f s i n g l e s t r a n d e d M 1 3 t e m p l a t e DNA. 90
3 . 1 7 C o m p l e m e n t a t i o n t e s t s on s i n g l e s t r a n d e d M 1 3 DNA. 91
3 . 1 8 P l a t i n g b a c t e r i o p h a g e lambda. 91
3 . 2 0 A u t o m a t i c e x c i s i o n of p h a g e m i d s f r o m
l a m b d a Z A P c l o n e s . 94
3.21 D N A s e q u e n c i n g b y the d i d e o x y c h a i n
t e r m i n a t i o n m ethod.
3 .21.1 S e q u e n c i n g s i n g l e s t r a n d e d M 1 3 t e m p l a t e s . 95
3 . 2 1 . 2 S e q u e n c i n g p l a s m i d DNA. 96
3 .22 O l i g o n u c l e o t i d e d i r e c t e d m u t a g e n e s i s .
3 .22.1 P h o s p h o r y l a t i n g s y n t h e t i c o l i g o n u c l e o t i d e s . 97
3 . 2 2 . 2 A n n e a l i n g o l i g o n u c l e o t i d e to t e m p l a t e . 98
3 . 2 2 . 3 E x t e n s i o n r e a c t i o n s . 98
3 . 2 2 . 4 S c r e e n i n g f or m u t a n t s . 99
3 .23 S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s .
3 .23.1 R u n n i n g p r o t e i n gels. 101
3 . 2 3 . 2 S t a i n i n g p r o t e i n g els w i t h C o o m a s s i e b lue. 101
3 . 2 3 . 3 F l u o r o g r a p h y of p r o t e i n gels. 1 02
3 .24 W e s t e r n b lots. 102
v i l i
3 . 2 6 G e n e r a t i o n a n d p u r i f i c a t i o n of a p o l y c l o n a l
a n t i s e r u m .
3 . 2 6 . 1 P u r i f i c a t i o n of a f u s i o n p r o t e i n p r o d u c e d in
E. coli for u se as a n a n t i g e n . 104
3 . 2 6 . 2 I m m u n i s a t i o n of r a bbit. 104
3 . 2 6 . 3 A f f i n i t y p u r i f i c a t i o n o f a n t i s e r u m . 105
3 . 2 7 S u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . 106
3 . 2 8 P r e p a r a t i o n o f n u c l e a r e x t r a c t f r o m X t c cells. 107
3 . 2 9 M e t h o d s for r a d i o l a b e l l i n g D N A a n d RNA.
3 . 2 9 . 1 N i c k t r a n s l a t i o n . 108
3 . 2 9 . 2 E n d l a b e l l i n g D N A w i t h ^ -3 2P - A T P e n d
T4 p o l y n u c l e o t i d e k i n a s e . 109
3 . 2 9 . 3 L a b e l l i n g D N A f r a g m e n t s b y e n d - f i l l i n g . 110
3 . 2 9 . 4 3 2 P l a b e l l e d s y n t h e t i c R N A pr o b e s . 1 10
3 . 3 0 P r e p a r a t i o n o f s y n t h e t i c R N A for m i c r o i n j e c t i o n
ix
3 . 3 1 P r e p a r a t i o n of p r o t e i n e x t r a c t f o r b a n d
s h i f t a s says.
3 . 3 1 . 1 F r o m X. l a e v i s o o c y t e s , e g g s a n d e m b r y o s . 113
3 . 3 1 . 2 F r o m a d u l t X. l a e v i s tis s u e s . 114
3 . 3 1 . 3 F r o m c u l t u r e d cells. 114
3 . 3 1 . 4 F r o m c o l i e x p r e s s i n g f u s i o n p r o t e i n s . 115
3 . 3 2 B a n d s h i f t assays.
3 . 3 2 . 1 P r e p a r a t i o n of pr o b e s . 1 15
RESULTS AND DISCUSSION,
C h a p t e r A: H o m o l o g u e s of h u m a n O c t - 1 o c c u r ± n the
X. l a e v l s g e n o m e , a n d in Xj. l a e v i s
o o c y t e RNA.
I n t r o d u c t i o n 117
A . l X_j_ l a e v i s g e n o m i c S o u t h e r n b lot. 117
A . 2 X. l a e v i s A+ s e l e c t e d R N A N o r t h e r n b lot. 118
C h a p t e r 5: I s o l a t i o n o f X e n o p u s l a e v i s O c t - 1 c D N A
c l o n e s f r o m a n o o c y t e lib r a r y .
5.1 L i b r a r y s c r e e n i n g . 120
5.2 A u t o m a t i c e x c i s i o n o f p r o s p e c t i v e O c t - 1 c D N A
c l o n e s f r o m the p h a g e v e c t o r a nd p r e l i m i n a r y
r e s t r i c t i o n a n a l y s i s . 1 2 1
5 .3 D e t a i l e d r e s t r i c t i o n a n a l y s i s of
c l o n e s 3, 6 a n d 16. 121
C h a p t e r 6: S e q u e n c e a n a l y s i s of O c t - 1 h o m o l o g u e s .
6.1 S e q u e n c i n g c l o n e s 3 a n d 16. 125
6.2 C o m p a r i s o n of X ^ l a e v i s O ct-1 p r o t e i n s e q u e n c e s
w i t h h u m a n a n d c h i c k e n O ct-1. 127
C h a p t e r 7: S o u t h e r n b l o t o f X. l a e v i s g e n o m i c D N A
p r o b e d w i t h P O U d o main, O c t - 1 s p e c i f i c
C h a p t e r 8: D e t e c t i o n o f X l - O c t - I A a n d X l - O c t - I B t r a n s c r i p t s i n l a e v i s o o c y t e R N A
b y R N a s e p r o t e c t i o n as s a y s .
I n t r o d u c t i o n . 132
8 . 1 S u b c l o n i n g o f a f r a g m e n t o f X l - O c t - I A i n t o a
t r a n s c r i p t i o n v e c t o r , f or u s e in the p r e p a r a t i o n
of s y n t h e t i c R N A p r o b e s . 133
8 . 2 The p r o b e d e t e c t s b o t h X l - O c t - I A a nd X l - O c t - I B
s y n t h e t i c RNA. 133
8 . 3 X l - O c t - I A a n d X l - O c t - I B t r a n s c r i p t s can be
d e t e c t e d i n X_j_ l a e v i s o o c y t e s . 134
C h a p t e r 9: E x p r e s s i o n o f X l - O c t - I A s y n t h e t i c R N A
in m i c r o - i n j e c t e d o o c y t e s . 136
C h a p t e r 10: P r e p a r a t i o n o f X l - O c t - I A f usion
p r o t e i n c o n s t r u c t s .
I n t r o d u c t i o n . 139
1 0 . 1 M a k i n g a Bgl II s i t e at the 5' e n d of X l - O c t - I A . 140
xii
C h a p t e r 11: P r o d u c t i o n of a n a n t i X l - O c t - I A
p o l y c l o n a l a n t i s e r u m .
I n t r o d u c t i o n . 143
1 1.1 M a k i n g an X l - O c t - I A f u s i o n p r o t e i n f o r use
a s a n a n t i g e n . 143
1 1 . 2 P r o d u c t i o n o f a p o l y c l o n a l a n t i s e r u m . 145
C h a p t e r 12: T he X e n o p u s l a e v i s O c t - 1 c D N A e n c o d e s a
f u n c t i o n a l o c t a m e r - b i n d i n g p r o t e i n . 1 47
C h a p t e r 13: T w o o c t a m e r - b i n d i n g p r o t e i n s c a n be
d e t e c t e d i n X e n o p u s l a e v i s o o c y t e ext r a c t .
1 3 . 1 O o c y t e s c o n t a i n O c t - 1 a n d a s e c o n d
o c t a m e r - b i n d i n g p r o t e i n . 150
1 3 . 2 A n a n t i b o d y a g a i n s t f u l l - l e n g t h h u m a n Oct-1
r e c o g n i s e s X e n o p u s l a e v i s O c t - 1 , b u t not O c t - R . 151
C h a p t e r 14: A c o m p a r i s o n of t h e b i n d i n g p r o p e r t i e s
a n d d i s t r i b u t i o n o f O c t - 1 a n d Oct-R.
1 4 . 1 O c t - 1 a n d O c t - R have d i f f e r e n t b i n d i n g
a f f i n i t i e s . 153
1 4 . 2 D i s t r i b u t i o n o f O ct-1 a n d O c t - R i n ti s s u e s
a n d e a r l y d e v e l o p m e n t . 155
1 4 . 3 O c t - R c a n n o t be d e t e c t e d in m o u s e L cells. 156
1 4 . 5 L e v e l s of O c t - 1 a n d O c t - R i n c e l l s in w h i c h D NA
s y n t h e s i s h a s b e e n i n h i b i t e d a n d in c e l l s w h i c h
h a v e b e e n s e r u m st a r v e d . 157
1 4 . 6 C o n c l u s i o n s a n d s p e c u l a t i o n r e g a r d i n g X e n o p u s
l a e v l s O c t - 1 a nd O c t - R b a s e d on the
a f f i n i t y / d i s t r i b u t i o n d a t a . 158
C h a p t e r 15: D o t h e o c t a m e r m o t i f a n d O c t - l / O c t - R
r e g u l a t e X e n o p u s l a e v i s h i s t o n e H 2 B g e n e s ?
I n t r o d u c t i o n . 171
1 5 . 1 P r e p a r a t i o n of a H 2 A - H 2 B e x p r e s s i o n c o n s t r u c t . 172
1 5 . 2 E x p r e s s i o n o f p H 2 A / B . e x p c a n be d e t e c t e d in
m i c r o i n j e c t e d oo c y t e s . 175
1 5 . 3 A n a t t e m p t to m o d u l a t e p H 2 A / B . e x p e x p r e s s i o n by
c o m p e t i t i o n w i t h oct f a c t o r b i n d i n g s i t e s . 175
1 5 . 4 M a k i n g a m u t a t i o n in the o c t a m e r m o t i f
a s s o c i a t e d w i t h the H 2 B g e n e of p H 2 A / B . e x p . 176
1 5 . 5 E f f e c t o f t he o c t a m e r m u t a t i o n on
H 2 A / B e x p r e s s i o n . 177
C h a p t e r l6: T h e a n t i O ct-1 p o l y c l o n a l a n t i s e r u m d e t e c t s p r o t e i n s o t h e r t h a n O c t - 1 on W e s t e r n blots.
1 6 . 1 T he a n t i O c t - 1 a n t i s e r u m s p e c i f i c a l l y d e t e c t s
xiv
16.2 O c t - 1 a n d the p r o t e i n s to w h i c h t h e a n t i O c t - 1
a n t i s e r u m r e a c t s o n a W e s t e r n b l o t c a n be
s e p a r a t e d o n a s u c r o s e g r adient.
16.3 D i s t r i b u t i o n o f O c t - 1 r e l a t e d p r o t e i n s in
o o g e n e s i s a n d e a r l y d e v e l o p m e n t .
16.4 L o c a t i o n o f O c t - 1 r e l a t e d p r o t e i n s i n t h e c ell.
C h a p t e r 17: G e n e r a l d i s c u s s i o n a nd c o n c l u s i o n s .
182
183
184
186
T a b l e o f f i g u r e s .
F i g u r e T i t l e . P r i o r
n u m b e r . to p a g e
1 T h e s t r u c t u r e o f h i s t o n e H 2 B g ene p r o m o t e r s . 1 0
2 A s e q u e n c e c o m p a r i s o n o f H 2 B b o xes. 1 1
3 A m o d e l f o r t r a n s c r i p t i o n a l a c t i v a t i o n b y
O c t - 1 a nd O c t - 2 . 26
4 T h e P O U d o m a i n . 3A
5 O c t - 1 b i n d s to d e g e n e r a t e o c t a m e r m o t i f s b y
a s s o c i a t i o n w i t h f l a n k i n g s e q u e n c e s . 37
6 H o m o l o g y b e t w e e n O c t - 1 a n d O ct-2 o u t s i d e the
P O U d o main. 38
7 S o u t h e r n b l o t o f X. l a e v i s g e n o m i c D N A p r o b e d
w i t h h u m a n O c t - 1 cDNA. 1 1 8
8 N o r t h e r n b l o t o f l a e v l a R N A p r o b e d w i t h
X V i
9 A t h i r d r o u n d s c r e e n of a p o s i t i v e c D N A c l o n e f rom
t h e X ^ l a e v i s o o c y t e c D N A l i b r a r y . 121
10 P a r t i a l r e s t r i c t i o n m a p s o f c l o n e s 3, 6 a n d 16 a l i g n e d to i n d i c a t e c o n s e r v a t i o n o f s o m e s i t e s . 1 23
11 C l o n e 6 d o e s n o t h y b r i d i s e t o a f r a g m e n t f r o m t h e 5' e nd o f h u m a n O c t - 1 . 124
12 S e q u e n c i n g s t r a t e g y f or c l o n e 3. 1 26
1 3 S e q u e n c i n g s t r a t e g y f or c l o n e 16. 1 26
14 C o m p l e t e n u c l e o t i d e s e q u e n c e of
c l o n e 3 ( X l - O c t - I A ) . 1 27
15 C o m p l e t e n u c l e o t i d e s e q u e n c e of
c l o n e 16 ( X l - O c t - I B ) . 127
16 N u c l e o t i d e s e q u e n c e a l i g n m e n t o f c l o n e 3
(X l - O c t - I A ) a n d c lone 16 ( X l - O c t - I B ) . 127
17 P r e d i c t e d a m i n o acid s e q u e n c e a l i g n m e n t o f h u m a n
18 A l t e r n a t e s p l i c i n g o ccurs at t he N ter m i n a l
e n d of h u m a n O c t - 1 . 129
19 S o u t h e r n b l o t o f X_^ laevis g e n o m i c D N A p r o b e d
w i t h POU d o m a i n , O ct-1 s p e c i f i c a n d O c t - 2
s p e c i f i c p r o b e s . 131
20 D e t e c t i o n o f X l - O c t - I A and X l - O c t - I B t r a n s c r i p t s
i n o o c y t e s b y R N a s e p r o t e c t i o n a s s a y s . 134
21 E x p r e s s i o n o f X l - O c t - I A s y n t h e t i c t r a n s c r i p t s
i n m i c r o - i n j e c t e d oocytes. 137
22 X l - O c t - l A s e q u e n c e c o n t a i n e d i n f u s i o n p r o t e i n
c o n s t r u c t s . 141
23 E x p r e s s i o n o f f u s i o n p r o t e i n s s u i t a b l e for
u s e as a n t i g e n s . 144
24 T h e anti O c t - 1 a n t i s e r u m d e t e c t s the c o l i
f u s i o n p r o t e i n a g a i n s t w h i c h it w a s r aised. 146
25 O c t - 1 s e q u e n c e c o n t a i n e d in f u s i o n p r o t e i n
c o n s t r u c t s a n d t h e i r b i n d i n g to a n o c t a m e r
x v i i i
26 B a n d s h i f t a s s a y s s h o w i n g t hat o o c y t e e x t r a c t
c o n t a i n s O c t - 1 a n d a s e c o n d o c t a m e r - b i n d i n g
p r o t e i n , O c t - R . 1 5 1
27 A n t i h u m a n O c t - 1 a n t i s e r u m r e a c t s w i t h X e n o p u s
l a e v i s O c t - 1 , b u t not O c t - R . 1 5 2
28 C o m p a r i s o n o f the b i n d i n g a f f i n i t i e s o f O ct-1
a n d O c t - R f o r d i f f e r e n t o c t a m e r - c o n t a i n i n g
o l i g o s u s i n g b a n d s hift a s s a y s . 1 5 4
29 D i s t r i b u t i o n s of O c t - 1 a n d O c t - R d e t e r m i n e d
b y b a n d s h i f t as s a y s . 1 5 5
30 B a n d s h i f t a s s a y s to s h o w that O c t - R c a n n o t be
d e t e c t e d i n m o u s e L c ell e x t r a c t . 1 5 6
31 L o c a t i o n o f O c t - 1 and O c t - R in X e n o p u s l a e v i s
o o c y t e s d e t e r m i n e d b y b a n d s h i f t a s s a y s . 1 5 7
32 B a n d s h i f t a s s a y s to d e t e r m i n e the l e v e l s of
O c t - 1 a n d O c t - R in X e n o p u s l a e v i s X t c c e l l s
t r e a t e d w i t h h y d r o x y u r e a o r s e r u m - s t a r v e d . 1 5 8
34 X 1 H W 2 3 i n t e r g e n i c r e g i o n a n d s t r u c t u r e of
p H 2 A / B . e x p . 173
35 p H 2 A / B . e x p is e x p r e s s e d in m i c r o - i n j e c t e d o o c y t e s . 176
36 E f f e c t o f c o m p e t i t i o n w i t h oct f a c t o r b i n d i n g
s i t e s o n p H 2 A / B . e x p e x p r e s s i o n i n ooc y t e s . 176
37 M u t a g e n e s i s of the o c t a m e r m o t i f a s s o c i a t e d
w i t h the H 2 B g e n e of p H 2 A / B . e x p . 177
38 A c o m p a r i s o n o f p H 2 A / B . e x p a n d p H 2 B . e x p - m u t
e x p r e s s i o n in ooc y t e s . 178
39 A n t i O c t - 1 a n t i s e r u m s p e c i f i c a l l y d e t e c t s two
p r o t e i n s in o o c y t e e x t r a c t . 181
40 T h e p r o t e i n to w h i c h the a n t i O c t - 1 a n t i s e r u m
r e a c t s a n d O c t - 1 c a n be s e p a r a t e d o n a s u c r o s e
g r a d i e n t . 183
41 D i s t r i b u t i o n of O ct-1 r e l a t e d p r o t e i n s in
o o g e n e s i s a n d e a r l y d e v e l o p m e n t . 184
X X
A c k n o w l e d g e m e n t s .
I am g r a t e f u l f o r t h e a s s i s t a n c e o f m y s u p e r v i s o r , B o b Old,
t h r o u g h o u t t he c o u r s e o f the p r o j e c t . A l s o , I w o u l d l i k e to
t h a n k G l e n S w e e n e y f o r a d v i c e a n d e n c o u r a g e m e n t .
M y t hanks a r e a l s o d u e to M a r c e l a V l a d for a s s i s t a n c e w i t h
m i c r o s c o p y , to L i z J o n e s for h e l p in t h e p r e p a r a t i o n o f a
p o l y c l o n a l a n t i b o d y , a n d to W i n s h i p H e r r f o r p r o v i s i o n of
m a t e r i a l s a nd u n p u b l i s h e d data.
I a c k n o w l e d g e R. W. O l d for p r o v i s i o n o f t h e p l a s m i d p R W 2 3
a nd a n anti Xi. l a e v i s T H R a n t i s e r u m , W. H e r r f o r p r o v i s i o n
o f the p l a s m i d p B S - O c t - l + a n d a n a n t i h u m a n O c t - 1 a n t i s e r u m ,
P. M a t t h i a s f o r p r o v i s i o n o f the h u m a n 0 c t - 2 A c DNA,
A. R. B rooks f o r p r o v i s i o n o f t he p l a s m i d a l b o n e A - 6 7 0 , and
J. S h u t t l e w o r t h f o r p r o v i s i o n o f an o o c y t e c D N A l i b r a r y .
F i n a n c i a l s u p p o r t w a s p r o v i d e d b y t he M e d i c a l R e s e a r c h
D e c l a r a t i o n .
A l l the r e s u l t s p r e s e n t e d in t his t h e s i s w e r e o b t a i n e d b y
the au t h o r , a p a r t f r o m t h o s e that a re s p e c i f i c a l l y i n d i c a t e d
in the text. A l l t he o o c y t e i n j e c t i o n s w e r e p e r f o r m e d b y
Bob Old.
A l l s o u r c e s o f i n f o r m a t i o n h a v e b e e n a c k n o w l e d g e d b y m e a n s
of r e f e r e n c e . N o n e o f the w o r k c o n t a i n e d i n t h i s t h e s i s h a s
X X il
Summary.
The ubiquitous human octamer-binding transcription factor, Oct-1, is believed to regulate the expression of a number of ubiquitously expressed genes. These include genes which are expressed throughout the cell-cycle (eg. snRNA genes) and histone H2B genes, whose expression is tightly coupled to nuclear DNA synthesis at S-phase of the cell-cycle.
I have isolated and completely sequenced two laevis homologues of Oct-1. The high degree of relatedness of the two homologues indicates that these are likely to be copies of the same gene, which arose during the theoretical genome duplication event in X. laevis evolution. X. laevis and human Oct-1 display strong evolutionary conservation (85% Identity over a stretch of 750 amino acids), which presumably means that X. laevis has a similar, if not identical function to human Oct-1. Homology between human and laevis does, however, break down shortly before the N terminal end, at a point where alternate splicing is known to occur in hunan Oct-1 (W. Herr, pers. comn.). The full length X. laevis cDNA clone which I have isolated may represent a novel alternately spliced form of Oct-1.
Two octamer-binding proteins have been identified (in band shift assays) in X. laevis oocyte, embryo and tissue extract. Oct-1, and a second, previously unidentified octamer-binding protein which has been termed Oct-R, for octamer-related. Oct-1 does not bind to a degenerate octamer motif most often seen in X^ laevis H2B promoters. Oct-R binds more strongly to this degenerate motif than the consensus motif, but only in the context of the H2B promoter, and does not bind either motif in another sequence context. This suggests that Oct-R may have a role in regulation of H2B transcription, although no direct evidence has been obtained.
Since Oct-1 is believed to stimulate the S-phase specific induction of histone H2B gene transcription the possibility that Oct-1 binding activity is cell-cycle regulated is of interest. X. laevis Oct-1 (and Oct-R) binding activity does not appear to be celT^cycle regulated.
Oct-1 and Oct-R are stored in the oocyte (partly in the cytoplasm), in an amount equivalent to at least 80 000 somatic cells. Histone protein and message are stored in the oocyte as part of the mechanism to provide enough histones to keep-up with the high rate of DNA synthesis in early Xenopus development. It is possible that histone gene transcription factors are stored for the same purpose.
By mutation of the octamer motif in the promoter of a L laevis histone H2B gene promoter I have tentatively concluded that the octamer motif is required for the expression of a H2B gene (independently of DNA synthesis) in the oocyte. The H2B gene occurs in association with a H2A gene, as part of a divergently expressed gene pair. The octamer motif may be required for the expression of both H2B and H2A genes. The degenerate octamer motif contained in this H2B promoter does not bind efficiently to Oct-1 in vitro, but binds well to Oct-R, indirectly suggesting that Oct-R is required for the expression of the H2B gene.
A b b r e v i a t i o n s
Ac a c e t a t e .
Ad a d e n o v i r u s .
A TP, dATP, a d e n o s i n e t r i p h o s p h a t e , d e o x y a d e n o s i n e
d d A T P t r i p h o s p h a t e , d i d e o x y a d e n o s i n e t r i p h o s p h a t e ,
bp b a s e pair.
B C I P A - c h l o r o - 3 - i n d o y l p h o s p h a t e .
c D N A c o m p l e m e n t a r y D N A .
Ci Curie.
C I A P c a l f i n t e s t i n a l a l k a l i n e p h o s p h a t a s e .
C T P , dCTP, c y t i d i n e t r i p h o s p h a t e , d e o x y c y t i d i n e
d d C T P t r i p h o s p h a t e , d i d e o x y c y t i d i n e t r i p h o s p h a t e .
D N A d e o x y r i b o n u c l e i c acid.
D N a s e d e o x y r i b o n u c l e a s e .
D T T d i t h i o t h r e i t o l .
d T T P , T h y m i d i n e t r i p h o s p h a t e , d e o x y t h y m i d i n e
d d T T P t r i p h o s p h a t e .
E D T A Et h y l e n e d i a m i n e t etra a c e t i c acid.
G H g r o w t h h o r m o n e .
G T P , dGTP, g u a n o s i n e t r i p h o s p h a t e , d e o x y g u a n o s i n e
d d G T P t r i p h o s p h a t e , d i d e o x y g u a n o s i n e t r i p h o s p h a t e
H A C0C I3 h e x a m i n o c o b a l t t r i c h l o r i d e .
H E P E S N - [ 2 - H y d r o x y e t h y l ] p i p e r a z i n e - N ' - [ 2 - e t h a n e
s u l p h o n i c a c i d ] .
x x i v
Ig I m m u n o g l o b u l i n
I g H i m m u n o g l o b u l i n h e a v y c hain.
Igk i m m u n o g l o b u l i n k a p p a l i g h t chain.
I P T G i s o p r o p y l - B - D - t h i o g a l a c t o s i d a s e .
Kb k i l o b a s e p airs.
K D k i l o d a l t o n s .
1 litre.
M E S 2 [ N - m o r p h o l i n o ] e t h a n e s u l p h o n i c a c i d
ml m i l l i l i t r e .
M O P S 3 - ( N - m o r p h o l i n o ) p r o p a n e s u l f o n i c a c i d .
M S 2 2 2 3 - a m i n o b e n z o i c a c i d e t h y l ester.
ng n a n o g r a m .
N B T n i t r o - b l u e t é t r a z o l i u m .
O R F o p e n r e a d i n g f rame.
p f u p l a q u e f o r m i n g u n its.
Pg pic o g r a m .
Pol p o l y m e r a s e .
Prl p r o l a c t i n .
R N A r i b o n u c l e i c a cid.
R N a s e r i b o n u c l é a s e .
rpra r e v o l u t i o n s p e r m i n u t e .
r R N A r i b o s o m a l RNA.
S Sve d b e r g .
S DS s o d i u m d o d e c y l s u l f a t e .
s n R N A s m a l l n u c l e a r RNA.
T C A t r i c h l o r o a c e t i c a c i d .
T r i s T r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e
t R N A t r a n s f e r RNA.
u g m i c r o g r a m .
ul m i c r o l i t r e .
U T P u r i d i n e t r i p h o s p h a t e .
v / v v o l u m e / v o l u m e .
w / v w e i g h t / v o l u m e .
X - g a l 5 - B r o m o - A - c h l o r o - 3 - i n d o y l - B - D -
I n t r o d u c t i o n
C H A P T E R 1: INTRODU C T I O N .
1.1 T h e O c t a m e r Paradox.
T h e o c t a m e r m o t i f v a s first s h o w n to be a t r a n s c r i p t i o n a l
c o n t r o l e l e m e n t i n the i m m u n o g l o b u l i n k a p p a c h a i n pro m o t e r
( F a l k n e r a n d Z a c h a u (1984)). The o c t a m e r m o t i f (ATTTGCAT)
o r i t s i n v e r s e c o m p l e m e n t w as s u b s e q u e n t l y r e c o g n i s e d as a
c o n s e r v e d p r o m o t e r / e n h a n c e r s e q u e n c e e l e m e n t i n v o l v e d in
the r e g u l a t i o n o f a r ange of g e n e s w i t h d i v e r s e p a t t e r n s of
e x p r e s s i o n . T h e s e g e n e s in c l u d e i m m u n o g l o b u l i n h e a v y and
l i g h t c h a i n g e n e s (the e x p r e s s i o n of w h i c h is r e s t r i c t e d to
B l y m p h o c y t e s ) , U S n R N A genes ( u b i q u i t o u s l y e x p r e s s e d ) and
h i s t o n e H 2 B g e n e s ( u b i q u i t o u s l y e x p r e s s e d at S - p h a s e of the
c e l l - c y c l e ) . T he o c t a m e r m o t i f a s s o c i a t e d w i t h t hese g enes
w a s s h o w n to b i n d a c o m m o n f a c t o r ( Sive a n d R o e d e r (1986)).
F o l l o w i n g this t h e r e w a s an e x p l o s i o n o f i n t e r e s t in h o w a
the s a m e motif, b i n d i n g a c o m m o n f a c t o r w a s a a b l e to
s p e c i f y d i v e r s e p a t t e r n s of g e n e e x p r e s s i o n . P r i o r to
r e c o g n i s i n g the o c t a m e r m o t i f as i m p o r t a n t t h e acc e p t e d
m o d e l w a s that the D N A b i n d i n g d o m a i n of a t r a n s c r i p t i o n
f a c t o r c o n f e r r e d the s p e c i f i c i t y of t r a n s c r i p t i o n a l
a c t i v a t i o n by a t t a c h i n g the a c t i v a t i o n d o m a i n to a
p a r t i c u l a r p r o m o t e r . T his m o d e l c a n n o t e x p l a i n h o w a c o m m o n
p r o m o t e r el e m e n t is a b l e to r e g u l a t e d i v e r s e l y e x p r e s s e d
g e n e s . T h e ' o ctamer paradox' h a s b e e n p a r t i a l l y r e s o l v e d b y
the i d e n t i f i c a t i o n of s everal f a c t o r s w h i c h b i n d the
o c t a m e r m o t i f s a s s o c i a t e d w i t h t hese g enes.
I n t he f e w y e a r s s i n c e t h e i r d i s c o v e r y , o c t a m e r - b i n d i n g
t r a n s c r i p t i o n f a c t o r s h a v e p r o b a b l y b e c o m e t he
t r a n s c r i p t i o n f a c t o r s a b o u t w h i c h m o s t is k n o w n . P r o p e r t i e s
w h i c h h a v e b e e n i n v e s t i g a t e d f or t he o c t a m e r - b i n d i n g
t r a n s c r i p t i o n f a c t o r s , a re a p p l i c a b l e to o t h e r f a c t o r s .
T h e s e i n c l u d e r e g u l a t i o n o f d i v e r s e l y e x p r e s s e d g e n e s ( also
s e e n w i t h , f o r e x a m p l e , N F - k B w h i c h r e g u l a t e s
i m m u n o g l o b u l i n g e n e s a n d a v a r i e t y o f g e n e s i n n o n - B c ells;
s e e L e n a r d o a n d B a l t i m o r e ( 1989)), f l e x i b l e b i n d i n g s ite
r e q u i r e m e n t s ( a l s o s e e n w i t h , for e x a m p l e , N F - k B a n d
C R E B / A T F ; s ee L e n a r d o a n d B a l t i m o r e (1 9 8 9 ) , J o n e s e t al
( 1 9 8 8 ) ) , a n d o c c u r r e n c e as a f a m i l y o f p r o t e i n s b i n d i n g to
t he s a m e s i t e s ( a l s o s e e n w i t h , f or e x a m p l e , f o s / j u n and
C R E B / A T F ; s ee Z i f f (1990)).
F o l l o w i n g t he c l o n i n g o c t a m e r - b i n d i n g t r a n s c r i p t i o n
f a c t o r s it b e c a m e a p p a r e n t that that t h e s e p r o t e i n s c o n t a i n
h o m e o b o x e s . T h e h o m e o b o x w a s i d e n t i f i e d a s a c o n s e r v e d
r e g i o n b e t w e e n D r o s o p h i l a h o m e o t i c p r o t e i n s ( G e h r i n g
( 1 9 8 7 ) ) . M u t a t i o n s in h o m e o t i c g e n e s t r a n s f o r m c e r t a i n
p a r t s , or a n e n t i r e b o d y s e g m e n t i n t o t h e c o r r e s p o n d i n g
s t r u c t u r e s o f a n o t h e r b o d y seg m e n t . S i n c e h o m e o b o x g e n e s
h a v e b e e n c l o n e d l a r g e l y b y c l a s s i c a l g e n e t i c a n a l y s i s or
b y h o m o l o g y to o t h e r h o m e o b o x g enes, t he t a r g e t g e n e s of
t h e s e p u t a t i v e t r a n s c r i p t i o n f a c t o r s a r e m u c h l e s s w e l l
d e f i n e d t h a n t h e t a r g e t s of o c t a m e r - b i n d i n g t r a n s c r i p t i o n
f a c t o r s . A s t u d y o f o c t a m e r - b i n d i n g t r a n s c r i p t i o n f a c t o r s
w i l l n o d o u b t p r o v i d e a n i n s i g h t as to h o w h o m e o b o x g e n e s
I n t r o d u c t i o n
in g e n e r a l r e g u l a t e t r a n s c r i p t i o n a n d t h e r e b y s p e c i f y
s e g m e n t a l i d e n t i t y . It is b e c o m i n g a p p a r e n t t hat s e v e r a l
D r o s o p h i l a h o m e o b o x p r o t e i n s b i n d to c o m m o n s i t e s ( r e v i e w e d
in B i g g i n a n d T i j a n ( 1989)), a n d f e a t u r e s d i s c o v e r e d for
the o c t a m e r - b i n d i n g t r a n s c r i p t i o n f a c t o r s , s u c h a s
i n t e r a c t i o n w i t h o t h e r p r o t e i n s v i a s p e c i f i c r e s i d u e s in
the h o m e o b o x , m a y be a p p l i c a b l e to the m e c h a n i s m s b y w h i c h
h o m e o b o x p r o t e i n s in g e n e r a l d i f f e r e n t i a l l y a c t i v a t e
t r a n s c r i p t i o n t h r o u g h c o m m o n b i n d i n g sites.
T h i s s e c t i o n d e s c r i b e s the g e n e s a s s o c i a t e d w i t h t h e
o c t a m e r m o t i f , a n d t h e i r d i v e r s e p a t t e r n s o f g e n e
e x p r e s s i o n f o r w h i c h the o c t a m e r m o t i f is n e c e s s a r y . As
a l r e a d y n o t e d , t h e ' o c t a m e r p a r a d o x ' h a s b e e n p a r t i a l l y
r e s o l v e d b y t he i d e n t i f i c a t i o n of s e v e r a l f a c t o r s ( u s i n g
b a n d s h i f t a s s a y s ) w h i c h b i n d to t he o c t a m e r m o t i f s
a s s o c i a t e d w i t h t h e s e g e n e s . H e r e , I d e s c r i b e b i n d i n g
s t u d i e s o n the b e s t c h a r a c t e r i s e d o c t a m e r - b i n d i n g p r o t e i n s
( O c t - 1 a n d O c t - 2 A / O c t - 2 B ) u s i n g c r u d e e x t r a c t s a n d p a r t -
p u r i f i e d o c t a m e r b i n d i n g p r o t e i n s in r e l a t i o n t o t he
e x p r e s s i o n o f t h e s e g e nes. S t u d i e s o n t he d i f f e r e n t i a l
a b i l i t i e s o f O c t - 1 a n d O c t - 2 ( i n c l u d i n g c l o n e d O c t - 1 and
O c t - 2 ) to a c t i v a t e t r a n s c r i p t i o n w i l l b e d i s c u s s e d i n m o r e
d e t a i l i n l a t e r s e c t i o n s , as w i l l s t u d i e s o n o t h e r o c t a m e r
b i n d i n g p r o t e i n s .
1 . 1 . 1 T h e o c t a m e r m o t i f a n d i m m u n o g l o b u l i n g e n e e x p r e s s i o n .
T r a n s c r i p t i o n o f i m m u n o g l o b u l i n g e n e s is b o t h t i s s u e
s p e c i f i c (in B l y m p h o c y t e s ) a n d d e v e l o p m e n t a l s t a g e
s p e c i f i c ( l o w e r l e v e l s o f e x p r e s s i o n i n e a r l y B c e l l
d e v e l o p m e n t ) . T h e t r a n s c r i p t i o n o f i m m u n o g l o b u l i n g e n e s is
c o n t r o l l e d b y b o t h p r o m o t e r a n d e n h a n c e r ( d o w n s t r e a m o f t h e
p r o m o t e r , w i t h i n i n t r o n s ) s e q u e n c e e l e m e n t s , w h i c h c o n t a i n
a c o m p l e x a r r a y o f f u n c t i o n a l l y a c t i v e t r a n s - a c t i n g f a c t o r
b i n d i n g s i t e s ( f o r r e c e n t r e v i e w s ee C a l a m e (19 8 9 ) ) .
O c t a m e r m o t i f s o c c u r w i t h i n the h e a v y c h a i n g e n e ( I g H )
p r o m o t e r , the I g H e n h a n c e r , the k a p p a l ight c h a i n g e n e
(IgK) p r o m o t e r , a n d t he I g K e n h a n c e r (a n o n - c o n s e n s u s
o c t a m e r ) ( C a l a m e (1 9 8 9 ) , C u r r i e a n d R o e d e r ( 1 9 8 9 ) ) . A n
e x t e n s i v e s e r i e s o f t r a n s f e c t i o n s t u d i e s ( c a r r i e d o u t b y
n u m e r o u s g r o u p s ) u s i n g c o n s t r u c t s c o n t a i n i n g Ig
p r o m o t e r s / e n h a n c e r s w i t h n o r m a l a n d m u t a t e d o c t a m e r m o t i f s
h a v e e x a m i n e d t h e r o l e of the o c t a m e r m o t i f in Ig
t r a n s c r i p t i o n . T h e o c t a m e r m o t i f in Ig p r o m o t e r s is
a b s o l u t e l y r e q u i r e d f or g e n e e x p r e s s i o n ( E a t o n a n d C a l a m e
( 1987), F a l k n e r a n d Z a c h a u ( 1 984)) a n d d e l e t i o n o f t h e
o c t a m e r m o t i f w i t h i n the I g H e n h a n c e r r e d u c e s g e n e
e x p r e s s i o n ( G e r s t e r e_t al^ ( 1 987), L e n a r d o ej^ a_l ( 1 9 8 7 ) ) .
T h e o c t a m e r is a l s o a d e t e r m i n a n t o f t he B c e l l s p e c i f i c
e x p r e s s i o n o f Ig g e n e s . S ix c o p i e s o f the I g H e n h a n c e r
o c t a m e r d o w n s t r e a m o f the /8-globin g e n e are a B c e l l
s p e c i f i c e n h a n c e r ( G e r s t e r et a l (1 9 8 7 ) ) a n d s y n t h e t i c
o c t a m e r m o t i f s i n s e r t e d i n t o t he r e n i n g e n e p r o m o t e r ( w h i c h
is n o r m a l l y e x p r e s s e d o n l y in h i g h l y s p e c i a l i s e d t i s s u e s )
l i n k e d to the y8- g l o b i n T A T A b o x c r e a t e a h i g h a c t i v i t y
-I n t r o d u c t i o n
l y m p h o i d s p e c i f i c p r o m o t e r ( D r e y f u s £ t al^ (1 9 8 7 ) ) , as do
o c t a m e r o l i g o n u c l e o t i d e s u p s t r e a m o f the £ - g l o b i n T A T A b o x
( W i r t h et al (1987)).
A v a r i e t y o f p r o t e i n s b i n d i n g to the o c t a m e r m o t i f h a v e
b e e n i d e n t i f i e d u s i n g b a n d s h i f t a s s a y s . T h e b e s t
c h a r a c t e r i s e d o c t a m e r - b i n d i n g p r o t e i n s a r e O c t - 1 ( S i n g h
et al ( 1986), L a n d o l f i e £ j»l ( 1 9 8 6 ) , G e r s t e r e_t ¿ 1 ( 1 987),
F l e t c h e r e £ al (1987) - a l s o k n o w n as O T F - 1 , 0 B P 1 0 0 , N F A 1
a n d N F I I I ) a n d 0 c t - 2 A ( L a n d o l f i e t al^ ( 1 9 8 6 ) , G e r s t e r e t al
( 1 9 8 7 ) , S t a u d t et al ( 1 9 8 6 ) , S c h e i d e r e i t et al ( 1987) -
a l s o k n o w n as O TF-2, N F A 2 , I g N F A ) , w h i c h b i n d
i n d i s t i n g u i s h a b l y to t he o c t a m e r m o t i f . O c t - 1 has b e e n
f o u n d in a l l c e l l t y p e s t e s t e d , a n d c o n s e q u e n t l y i m p l i c a t e d
i n t he r e g u l a t i o n of u b i q u i t o u s l y e x p r e s s e d o c t a m e r -
c o n t a i n i n g g e n e s . 0 c t - 2 A is p r e d o m i n a n t l y e x p r e s s e d in
l y m p h o i d t i s s u e s ( e x p r e s s i o n is a l s o d e t e c t e d in the
c e n t r a l n e r v o u s sy s t e m , a n d o t h e r t i s s u e s ) , a n d is
c o n s e q u e n t l y i m p l i c a t e d in t he r e g u l a t i o n o f Ig g e n e s . A
s e c o n d l y m p h o i d s p e c i f i c o c t a m e r - b i n d i n g p r o t e i n h a s b e e n
i d e n t i f i e d ( 0 c t - 2 B ) ( G e r s t e r e £ a_l (1 9 8 7 ) , S c h r e i b e r et al
( 1 9 8 8 ) ) b y b a n d s h i f t a s s a y s . 0 c t - 2 A / 0 c t - 2 B h a v e
s u b s e q u e n t l y b e e n s h o w n to b e d e r i v e d f r o m t he s a m e g e n e b y
d i f f e r e n t i a l s p l i c i n g ( H a t z o p o u l o s et al (19 9 0 ) ) . 0 c t - 2 A
i t s e l f m a y c o n s i s t o f t h r e e p r o t e i n s p e c i e s ( S c h e i d e r e i t
e ^ al^ (19 8 7 ) ) . S u b s e q u e n t l a b e l l i n g o f c e l l s t r a n s f e c t e d
w i t h c l o n e d O c t - 2 w i t h ^ S a n d h a s s h o w n t hat d i f f e r e n t
p h o s p h o r y l a t e d f o r m s o f 0 c t - 2 A e x i s t ( T a n a k a a n d H e r r
-(19 9 0 ) ) , t h i s m a y e x p l a i n the m u l t i p l e f o r m s o f 0 c t - 2 A
i d e n t i f i e d b y p u r i f i c a t i o n o f t he p r o t e i n . T h e o b s e r v a t i o n
t h a t b a c t e r i a l l i p o p o l y s a c c h a r i d e , w h i c h a c t i v a t e s
i m m u n o g l o b u l i n e x p r e s s i o n in B c e l l l i n e s , i n d u c e s O c t - 2
( b u t n ot O c t - 1 ) b i n d i n g a c t i v i t y ( S t a u d t et al ( 1 9 8 6 ) ) a dds
w e i g h t to the t h e o r y that O c t - 2 is i n v o l v e d in Ig g e n e
t r a n s c r i p t i o n .
T h e b i n d i n g p a t t e r n s o f e x t r a c t f r o m l y m p h o i d a n d H e L a
c e l l s to o c t a m e r - c o n t a i n i n g D N A f r a g m e n t s o r i g i n a l l y
d e s c r i b e d d i d n o t a l w a y s c o n s i s t s i m p l y o f t h r e e b a n d s
(O c t - 1 , 0 c t - 2 A , 0 c t - 2 B ) p r o b a b l y b e c a u s e t h e o r i g i n a l b a n d
s h i f t p r o b e s w e r e r e s t r i c t i o n f r a g m e n t s w i t h o t h e r b i n d i n g
s i t e s ( p a r t i c u l a r l y L a n d o l f i e_t aJL ( 1 9 8 6 ) , w h o s e p r o b e
c o n t a i n e d a n h e p t a m e r m o t i f , n o t t h e n r e c o g n i s e d as an
o c t a m e r b i n d i n g s i t e - s ee l a t e r ) , a n d b e c a u s e s o m e g r o u p s
d i d not d e t e c t O c t - 2 B in t h e i r e x t r a c t s ( p r o b a b l y a
t e c h n i c a l p r o b l e m ) .
T h e h e p t a m e r m o t i f is a c o n s e r v e d s e q u e n c e e l e m e n t
a d j a c e n t to t he o c t a m e r in t he I g H p r o m o t e r ( E a t o n a n d
C a l a m e ( 1 9 8 7 ) ) , w h i c h c o n t r i b u t e s to t h e a c t i v i t y o f the
p r o m o t e r . B o t h O c t - 1 a n d 0 c t - 2 A b i n d to the h e p t a m e r m o tif,
b u t o n l y w h e n the i n t a c t o c t a m e r m o t i f is p r e s e n t a n d
o c c u p i e d ( L a n d o l f i et al (1 9 8 8 ) , P o e l l i n g e r a n d R o e d e r
( 1 9 8 9 ) ) . B i n d i n g s t u d i e s w i t h c l o n e d 0 c t - 2 A h a v e s h o w n t hat
t he s ame r e g i o n o f 0 c t - 2 A is r e s p o n s i b l e f o r b i n d i n g to the
o c t a m e r a n d t he h e p t a m e r (a h i g h l y d e g e n e r a t e o c t a m e r
m o t i f ) ( L e B o w i t z ejt al ( 1 9 8 9 ) ) . 0 c t - 2 B a l s o b i n d s i n a
\
I n t r o d u c t i o n
s i m i l a r c o o p e r a t i v e f a s h i o n ( K e m l e r e £ a l ( 1 9 8 9 ) ) . T h i s
f e a t u r e m a y h a v e a r o l e in the a c t i v a t i o n o f I g H g enes
b e f o r e Igk g e n e s . P r e B cell l i n e s h a v e b e e n s h o w n to
c o n t a i n l e s s 0 c t - 2 A ( S t a u d t
et_
£ l ( 1 9 8 8 ) ) , a n d the c o o p e r a t i v e b i n d i n g m a y f a v o u r the a c t i v a t i o n o f I g H i n thep r e s e n c e o f l o w l e v e l s of 0c t - 2 A .
T h e r e is n o s i m p l e r e l a t i o n s h i p b e t w e e n I g e x p r e s s i o n a nd
O c t - 2 e x p r e s s i o n . O c t - 2 e x p r e s s i o n is d e t e c t e d (at l o w e r
l e v e l s ) in c e l l s w h i c h do n o t e x p r e s s I g g e n e s (see s e c t i o n
1 . 3 . 1 ) . The r o l e o f O c t - 2 i n c e l l s n o t e x p r e s s i n g Ig g e n e s
i s not c l e a r . A l s o , I g H e x p r e s s i o n o c c u r s i n c e l l l i n e s
w h i c h do n o t e x p r e s s O c t - 2 ( C o c k e r i l l a n d K l i n k e n (19 9 0 ) ) .
T h i s s e r v e s to p o i n t to the i n v o l v e m e n t o f o t h e r f a c t o r s in
t h e c o n t r o l o f Ig g e n e e x p r e s s i o n .
A n o t h e r a s p e c t o f Ig g ene e x p r e s s i o n is e x t i n c t i o n in
h y b r i d c ell f u s i o n s . G e n e s a c t i v a t e d b y t h e I g H e n h a n c e r
a r e i n a c t i v a t e d o n t h e f u s i o n of B c e l l s w i t h a T c e l l
l y m p h o m a c e l l l ine. L i k e w i s e , g e n e s a c t i v a t e d b y a
m u l t i m e r i s e d o c t a m e r ( w h i c h is a B c e l l s p e c i f i c e n h a n c e r )
a r e i n a c t i v a t e d . T h i s d o e s n o t c o r r e s p o n d t o the l o s s of
0 c t - 2 b i n d i n g a c t i v i t y (Yu e_t al ( 1 9 8 9 ) ) . I n a c t i v a t i o n is
d o m i n a n t to a c t i v a t i o n by the SVAO e n h a n c e r , a n d so
p o s s i b l y n o t d u e to the l o s s o f an I g H e n h a n c e r
t r a n s a c t i v a t o r . T h i s s u g g e s t s that T c e l l s c o n t a i n a
n e g a t i v e r e g u l a t o r , w h i c h a s s o c i a t e s w i t h t h e o c t a m e r
m o t i f . T h e r e is o t h e r e v i d e n c e of n e g a t i v e r e g u l a t o r s
a c t i n g v ia t h e o c t a m e r m o t i f ( L e n a r d o e £ a^. (1 9 8 9 ) ) . An
-o c t a m e r - c -o n t a i n i n g f r a g m e n t f r -o m the I gH e n h a n c e r is a c t i v e
i n n o n - l y m p h o i d c e l l s , but i n a c t i v e in the F9 ( e m b r y o n a l
c a r c i n o m a ) c ell line. H o w e v e r , m u t a t i o n i n t he o c t a m e r
r e n d e r s t h e f r a g m e n t an a c t i v e e n h a n c e r . A n F9 s p e c i f i c
o c t a m e r b i n d i n g a c t i v i t y ( N F A - 3 ) h a s b e e n d e t e c t e d , a n d is
a c a n d i d a t e for the n e g a t i v e r e g u l a t o r . H o w e v e r e x t i n c t i o n
o f the I g k e n h a n c e r on f u s i o n o f a m y e l o m a c e l l l i n e w i t h a
f i b r o b l a s t c ell l i n e is a s s o c i a t e d w i t h d e c r e a s e d O c t - 2
b i n d i n g a c t i v i t y ( a n d m e s s a g e ) ( B e r g m a n
eJL
al. ( 1 9 9 0 ) ) . S i m i l a r l y , B c e l l s s t a b l y t r a n s f e c t e d w i t h a c o n s t r u c tc o n t a i n i n g the Igk p r o m o t e r u p s t r e a m of a y ^ - g l o b i n r e p o r t e r
g e n e s h o w e x t i n c t i o n o f the r e p o r t e r g ene o n f u s i o n w i t h a
f i b r o b l a s t c ell l i n e ( J u n k e r e t al ( 1990)). T h i s e x t i n c t i o n
is d e p e n d e n t on the o c t a m e r m o t i f (if r e p l a c e d b y N F 1 or
Spl b i n d i n g sites t h e r e is n o e x t i n c t i o n ) . T h e e x t i n c t i o n
is o v e r c o m e b y t r a n s f e c t i o n w i t h c l o n e d 0 c t - 2 A , a n d
e x t i n c t i o n is a s s o c i a t e d w i t h t h e l o s s of 0 c t - 2 A / B m e s s a g e
a n d p r o t e i n .
1 . 1 . 2 T h e o c t a m e r m o t i f a n d h i s t o n e H 2 B g e n e e x p r e s s i o n .
T he m a j o r class o f h i s t o n e g e n e s in e u k a r y o t e s is
r e p l i c a t i o n d e p e n d e n t . B i o s y n t h e s i s is c l o s e l y l i n k e d to
n u c l e a r D N A s y n t h e s i s at S - p h a s e o f the c e l l - c y c l e . A
s u b c l a s s o f h i s t o n e g e n e s s h o w a l o w l e v e l o f c o n s t i t u t i v e
e x p r e s s i o n . The r e g u l a t i o n o f t h e r e i t e r a t e d r e p l i c a t i o n -
d e p e n d e n t h i s t o n e g e n e s o c c u r s at p o s t t r a n s c r i p t i o n a l
I n t r o d u c t i o n
f o r m a t i o n ) a n d t r a n s c r i p t i o n a l l e v e l s ( for r e v i e w s ee
S c h u m p e r l i ( 1 9 8 8 ) ) . The t r a n s c r i p t i o n a l r e g u l a t i o n o f
h i s t o n e s u b t y p e s in h i g h e r e u k a r y o t e s s e e m s to d e p e n d o n
s u b t y p e - s p e c i f i c p r o m o t e r e l e m e n t s . A s I w i l l d e s c r i b e in
m o r e d e t a i l b e l o w , H2B g e n e s h a v e a n o c t a m e r m o t i f i n t h e
p r o m o t e r r e q u i r e d for the S - p h a s e s t i m u l a t i o n o f
t r a n s c r i p t i o n . HI g enes h a v e a s u b t y p e - s p e c i f i c c o n s e n s u s
e l e m e n t , s h o w n b y m u t a t i o n a l a n a l y s i s to be r e q u i r e d f o r
the S - p h a s e s p e c i f i c s t i m u l a t i o n o f H i e x p r e s s i o n in
t r a n s f e c t e d H e L a c e l l s ( D a l t o n a n d W e l l s ( 1 9 8 8 ) ) . T h e l e v e l
o f a f a c t o r b i n d i n g to t h i s e l e m e n t i s i n c r e a s e d a t S - p h a s e
o f the c e l l - c y c l e (D a l t o n a n d W e l l s ( 1 9 8 8 A ) ) . T h e
a r r a n g e m e n t o f c i s - a c t i n g s i t e s in H 4 g e n e s is m u c h m o r e
c o m p l e x (see D a i l e y et al ( 1988) a n d r e f e r e n c e s t h e r e i n ) ,
b u t a s u b t y p e - s p e c i f i c c o n s e n s u s e l e m e n t h a s b e e n
i d e n t i f i e d a n d a b i n d i n g f a c t o r p u r i f i e d . It is n o t c l e a r
f r o m a n y m u t a t i o n a l a n a l y s i s t hat t h i s e l e m e n t is r e q u i r e d
f o r the S - p h a s e s p e c i f i c s t i m u l a t i o n o f t r a n s c r i p t i o n i n
v i v o , b u t t h i s f a c t o r is a b l e to a c t i v a t e t r a n s c r i p t i o n
f r o m p r o m o t e r s c o n t a i n i n g the c o n s e n s u s e l e m e n t _iii v i t r o .
N o c l e a r c o n s e n s u s e l e m e n t h a s b e e n d e s c r i b e d f o r h i s t o n e
H 3 p r o m o t e r s , b u t s e q u e n c e s r e q u i r e d f o r the S - p h a s e
s p e c i f i c s t i m u l a t i o n o f t r a n s c r i p t i o n of a h a m s t e r H 3 g e n e
h a v e b e e n i d e n t i f i e d , a n d f a c t o r s b i n d i n g to t h e s e
s e q u e n c e s s h o w e n h a n c e d l e v e l s at S - p h a s e of t he c e l l - c y c l e
( A r t i s h e v s k y e t al^ (1987)).
A n o t h e r a s p e c t o f h i s t o n e g e n e r e g u l a t i o n is t he
-T he s t r u c t u r e of h i s t o n e H 2 B g e n e p r o m o t e r s .
c o o r d i n a t e e x p r e s s i o n at S - p h a s e o f the c e l l - c y c l e . If
s u b t y p e - s p e c i f i c e l e m e n t s a r e r e s p o n s i b l e for the S - p h a s e
s p e c i f i c s t i m u l a t i o n o f t r a n s c r i p t i o n , t h e r e m u s t b e a
c o m m o n m e c h a n i s m f o r t h e e x p r e s s i o n / m o d i f i c a t i o n o f b i n d i n g
f a c t o r s ( i n c l u d i n g O c t - 1 , if it r e g u l a t e s h i s t o n e H 2 B
g e n e s ) . H i s t o n e g e n e s a r e m u l t i c o p y , a n d o c c u r in c l u s t e r s .
In b i r d s a n d m a m m a l s t h e c l u s t e r s are ' r andomly' o r g a n i s e d ,
b ut in a m p h i b i a n s a n d l o w e r e u k a r y o t e s t h e h i s t o n e g e n e s
a r e g e n e r a l l y o r g a n i s e d i n t o q u i n t e t s o f the 5 s u b t y p e s ,
a n d in s o m e c a s e s t he q u i n t e t s a r e s t r i c t l y o r d e r e d (for
r e v i e w s ee O l d a n d W o o d l a n d (1984)). T h e q u i n t e t
o r g a n i s a t i o n w a s o n c e t h o u g h t to h a v e s o m e r e g u l a t o r y
s i g n i f i c a n c e ( p e r h a p s i n c o o r d i n a t e c o n t r o l ) , b u t t h e r e is
n o e v i d e n c e f or t his. I n s t e a d , t he q u i n t e t o r g a n i s a t i o n may
h a v e a n e v o l u t i o n a r y s i g n i f i c a n c e a s s o c i a t e d w i t h t he
p r e s s u r e to m a i n t a i n e q u a l n u m b e r s o f h i s t o n e g e n e s u b t y p e s
o n m u l t i p l i c a t i o n .
T he s t r u c t u r e o f t y p i c a l h uman, c h i c k e n a n d X e n o p u s laevis
h i s t o n e H 2 B g ene p r o m o t e r s is i l l u s t r a t e d in f i g u r e 1. In
c h i c k e n a n d X e n o p u s l a e v i s H 2 B g e n e s g e n e r a l l y o c c u r
a s s o c i a t e d w i t h H 2 A g e n e s as p a r t of a d i v e r g e n t l y
e x p r e s s e d g e n e p a i r . A n o c t a m e r m o t i f o c c u r s in t h e H 2 B
g e n e p r o m o t e r , as t he c o r e of a l o n g e r c o n s e n s u s s e q u e n c e
k n o w n as the H 2 B box.
T r a n s f e c t i o n s t u d i e s w i t h r e p o r t e r c o n s t r u c t s c o n t a i n i n g a
w i l d - t y p e , a n d m u t a t i o n s o f a, h u m a n h i s t o n e H 2 B g e n e
p r o m o t e r h a v e r e v e a l e d the f o l l o w i n g f a c t s ( L a B e l l a e ^ al
-A s e q u e n c e c o m p a r i s o n of H 2 B boxes.
H 2 B b o x e s f r o m X. l a e v i s . c h i c k e n , m o u s e a n d h u m a n H 2 B gene p r o m o t e r s a r e aTT g n e d . A s p e c i e s c o n s e n s u s seq u e n c e is d e t e r m i n e d f o r l aevis a n d ch i c k e n , s ince the s e q u e n c e of s e v e r a l H 2 B p r o m o t e r s is a v a i l a b l e . B e n e a t h the c o n s e n s u s s e q u e n c e the n u m b e r of t i m e s a p a r t i c u l a r b a s e o c c u r s is s h o w n . M a k i n g the a s s u m p t i o n t hat the s i n g l e m o u s e and h u m a n s e q u e n c e s a v a i l a b l e a r e r e p r e s e n t a t i v e , an o v e r a l l c o n s e n s u s is d e t e r m i n e d f r o m t hese s e q u e n c e s a n d the c h i c k e n a n d
X^_
l a e v i s c o n s e n s u s sequ e n c e s . S e q u e n c e s are f r o m L a B e l l a et al (T988), S t u r m et al (1988), P e r r y et al^ ( 1 9 8 5 ) , M o o r m a n et al (1 9 8 2 ) , W e l l s ’ T T 9 8 6 ) , Z h o n g et a T ( 1 9 8 3 ) a n d A l d r i H g e “ C l 9 8 6 ) .S P E C I E S C L O N E
X. l aevis X 1 H W 2 3 X 1HW11 X1H1 X 1HW8 X 1H3 XlHhil X 1HW7 X 1 H W 2 8
C O N S E N S U S OUT O F 8
C h i c k e n p C H 3 . 3 E P C H 2 2 . 0 B p C H 3 . 5 E p C H l l . O E L p C H l l . O E R C H I - 1 0
C O N S E N S U S O U T O F 6
H u m a n p H h 4 c
M o u s e p M H 2 B
O V E R A L L C O N S E N S U S
O U T O F 4
O C T A M E R
C T G C C T C G T T T G C A T G G G A C T G C C T C G T T T G C A T G G G A C T G C C T C G T T T G C A T G G G A C A G C C T C G T T T G C A T G C G A G C C A G T G C T T T A C A T G G G A C T G C C T T A T T T G C A T G G G A C T A C C T T A T T T G C A T G G G A C T G C C T T A T T T G C A T G G G A
C T G C C T C G T T T G C A T G G G A 7 6 6 7 7 8 4 4 8 8 8 7 8 8 8 8 7 8 8
G C T T C T C A T T T G C A T A G A G T C T C C T A A T T T G C A T A C C G G C T G C T G C T T T G C A T A G C G G C T G C T G C T T T G C A T A G C G G A A C C T A A T C T G C A T A C A G C C T C T G T A T T T G C A T A A C A
G C T C C T N A T T T G C A T A G C G 4 4 5 3 5 5 - 4 6 5 6 6 6 6 6 6 3 4 5
TCACCTT ATTTGCAT AACG
G C T T G A C G T T T G C A G A C T C
G C T C C T C ¿ T T T G C A T A G G C
I n t r o d u c t i o n
( 1 9 8 8 ) ) . T h e d i r e c t repeats, hexaraer a n d C C A A T b o x c a n a ll
be d e l e t e d w i t h o u t a f f e c t i n g t h e s t i m u l a t i o n of
t r a n s c r i p t i o n o n e n t r y into S - p h a s e o f the c e l l - c y c l e
( w h i c h is n o r m a l l y a p p r o x i m a t e l y 5 fold). M u t a t i o n of the
o c t a m e r , h o w e v e r , c o m p l e t e l y a b o l i s h e s t h i s s t i m u l a t i o n .
T h e d i r e c t r e p e a t s a n d C C A A T b o x a r e r e q u i r e d f o r b o t h
b a s a l and S - p h a s e l e v e l s of e x p r e s s i o n . M u t a t i o n / d e l e t i o n
o f t h e s e e l e m e n t s g i v e s a l o w e r b a s a l a c t i v i t y , w h i c h is
s t i m u l a t e d a p p r o x i m a t e l y 5 - f o l d at S - p h a s e , g i v i n g a l o w e r
S - p h a s e l e v e l o f a c t i v i t y . T h e o c t a m e r is n o t r e q u i r e d f o r
b a s a l e x p r e s s i o n . T h e c o n s e r v e d h e x a m e r ( w h i c h m a k e s a
s m a l l c o n t r i b u t i o n to e x p r e s s i o n in in v i t r o a s s a y s (see
S i v e e £ al ( 1 9 8 6 ) ) h a s no e f f e c t o n e x p r e s s i o n i n t hese
t r a n s f e c t i o n a s s a y s .
T h e o c t a m e r m o t i f h a s b e e n s h o w n to be r e q u i r e d f o r the
s t e a d y s t a t e l e v e l s of c h i c k e n H 2 B a n d H 2 A t r a n s c r i p t i o n in
t r a n s f e c t e d c e l l s ( S t u r m et al ( 1 988)). H e r e the g e n e s
o c c u r as a d i v e r g e n t l y e x p r e s s e d pair, a n d o c t a m e r
m u t a t i o n s d e c r e a s e the e x p r e s s i o n o f b o t h g e n e s . S e p a r a t i o n
o f t he t wo g e n e s r e d u c e s e x p r e s s i o n of b o t h g e n e s m o r e t han
t h e o c t a m e r m u t a t i o n , a nd d e l e t i o n o f the C C A A T b o x m o s t
p r o x i m a l to t he H 2 B g ene d e c r e a s e s e x p r e s s i o n o f both,
g e n e s . C o n s e q u e n t l y the g e n e s m a y h a v e o t h e r p r o m o t e r
e l e m e n t s i n c o m m o n .
A s a l r e a d y n o t e d , in H 2B g e n e p r o m o t e r s the o c t a m e r o c c u r s
as t he c o r e o f a l o n g e r c o n s e n s u s s e q u e n c e , k n o w n as the
H 2 B b o x ( see f i g u r e 2, s e q u e n c e s f r o m L a B e l l a e £ al_ ( 1 988),
-S t u r m al^ ( 1 988), P e r r y <^t al^ (1985), M o o r m a n et al
( 1 9 8 2 ) , W e l l s ( 1 9 8 6 ) , Z h o n g et al ( 1 9 8 3 ) , A l d r i d g e (1 9 8 6 ) ) .
T h e H 2 B b o x is a l w a y s r e f e r r e d to as b e i n g c o n s e r v e d
b e t w e e n s p e c i e s . T h e o c t a m e r ( A T T T G C A T ) i s w e l l c o n s e r v e d
b e t w e e n H 2 B p r o m o t e r s w i t h i n a p a r t i c u l a r s p e c i e s a n d
b e t w e e n s p e c i e s , a l t h o u g h d e g e n e r a t e o c t a m e r m o t i f s o c c u r .
T h e s e q u e n c e G T T T G C A T is t he m o s t c o m m o n d e g e n e r a t e o c t a m e r
m o t i f , a n d G T T T G C A T is the m o s t c o m m o n o c t a m e r m o t i f i n
X e n o p u s l a e v i s h i s t o n e H 2 B g e n e p r o m o t e r s . Ho w e v e r ,
c o n s e r v a t i o n of s e q u e n c e s f l a n k i n g the o c t a m e r is m o s t
s t r i k i n g b e t w e e n g e n e s w i t h i n a p a r t i c u l a r sp e c i e s . T h i s is
p a r t i c u l a r l y true f o r X e n o p u s l a e v i s . w h e r e s t r o n g
c o n s e r v a t i o n is s e e n o v e r a s t r e t c h o f 19 bases.
C o n s e r v a t i o n o f s e q u e n c e s f l a n k i n g t he o c t a m e r b e t w e e n
s p e c i e s is, h o w e v e r , n o t e x t e n s i v e . In f i g u r e 2, Xz. l a e v i s
( f r o m 8 s e q u e n c e s ) a n d c h i c k e n ( from 6 s e q u e n c e s ) H 2 B b o x
c o n s e n s u s s e q u e n c e s h a v e b e e n d e t e r m i n e d . T h e s e s e q u e n c e s
a r e n o t w e l l m a t c h e d o u t s i d e the o c t a m e r , a n d d o n o t m a t c h
w e l l to t h e h u m a n a n d m o u s e H 2 B b o x e s o u t s i d e t he o c t a m e r .
O n l y o n e h u m a n a n d o n e m o u s e H 2 B b o x s e q u e n c e is a v a i l a b l e ,
a n d m a k i n g the a s s u m p t i o n t h a t t hese s e q u e n c e s are
r e p r e s e n t a t i v e , a n o v e r a l l c o n s e n s u s f r o m t hese s e q u e n c e s
a n d the c h i c k e n a n d X^_ l a e v i s c o n s e n s u s s e q u e n c e s is s h o w n
i n f i g u r e 2.
H u m a n ( S i v e a n d R o e d e r ( 1 9 8 6 ) ) , h a m s t e r ( Ito et al
( 1 9 8 9 ) ) , a n d c h i c k e n ( D a l t o n a n d W e l l s ( 1 9 8 8 A ) ) O c t - 1 h a v e
b e e n s h o w n to b i n d to the H 2 B o c t a m e r , a n d I h a v e s h o w n
