1993 au molecular cloning and sequence analysis of the cdna for ancrod c. rhodostoma
TRANSCRIPT
-
8/17/2019 1993 Au Molecular Cloning and Sequence Analysis of the CDNA for Ancrod C. Rhodostoma
1/4
Biochem.
J. (1993) 294
387 390 (Printed
in Great
Britain)
M o l e c u l a r
c l o n i n g
a n d
s e q u e n c e
a n a l y s i s
o f t h e
c D N A
f o r
a n c r o d ,
a
t h r o m b i n - l i k e
enzyme f r o m
t h e
venom o f C a l l o s e l a s m a
r h o d o s t o m a
L o - C h u n A U , * t §
S h w u - B i n L I N , t
J e n g - S h e n g
C H O U , 4
G o - W a i
T E H , * K u o - J u i
C H A N G t
a n d
C h i a - M i n
S H I H t
* D e p a r t m e n t o f M e d i c a l
R e s e a r c h , V e t e r a n s G e n e r a l
H o s p i t a l - T a i p e i , T a i p e i , t S c h o o l
o f M e d i c a l
T e c h n o l o g y ,
N a t i o n a l
Y a n g M i n g
M e d i c a l
C o l l e g e ,
T a i p e i ,
a n d
t G r a d u a t e
I n s t i t u t e
o f M e d i c a l
T e c h n o l o g y , D e p a r t m e n t
o f
C l i n i c a l
P a t h o l o g y , C o l l e g e
o f
M e d i c i n e ,
N a t i o n a l T a i w a n
U n i v e r s i t y ,
T a i p e i , T a i w a n ,
R e p u b l i c
o f
C h i n a
T h e
1 . 5 4 k b
cDNA
f o r
a n c r o d ,
a
t h r o m b i n - l i k e
enzyme,
wa s
c l o n e d f r o m a AZAP
cDNA
l i b r a r y d e r i v e d f r o m t h e venom
g l a n d s o f C a l l o s e l a s m a
( A g k i s t r o d o n ) r h o d o s t o m a . T h e
cDNA
sequence r e v e a l s t h a t
a n c r o d
i s
s y n t h e s i z e d
as
a
pre-zymogen o f
2 5 8 a m i n o a c i d s , i n c l u d i n g
a p u t a t i v e
s e c r e t o r y
p e p t i d e o f
1 8
a m i n o a c i d s a n d
a
p r o p o s e d zymogen p e p t i d e o f 6 a m i n o - a c i d
r e s i d u e s . T h e a m i n o - a c i d
sequence
o f t h e
p r e d i c t e d
a c t i v e
f o r m
o f
t h e e n z y m e
e x h i b i t s
a
h i g h
d e g r e e
o f
sequence s i m i l a r i t y
t o
t h o s e o f m a m m a l i a n
s e r i n e
p r o t e a s e s ( t r y p s i n a n d
p a n c r e a t i c
k a l l i k r e i n ) a n d o t h e r
t h r o m b i n - l i k e
e n z y m e s ( b a t r o x o b i n
a n d
I N T R O D U C T I O N
S n a k e venoms
a f f e c t
b l o o d
c o a g u l a t i o n
a n d
p l a t e l e t
f u n c t i o n s i n
a
c o m p l e x
manner
[ 1 ] .
T h e
venom o f
t h e
s n a k e C a l l o s e l a s m a
r h o d o s t o m a
( f o r m e r l y k n o w n as
A g k i s t r o d o n
r h o d o s t o m a )
c o n t a i n s
b o t h
p l a t e l e t - a c t i v a t i n g
a n d
p l a t e l e t - i n h i b i t o r y
com-
p o n e n t s ,
a
h a e m o r r h a g i c p r o t e i n a n d
a
t h r o m b i n - l i k e
e n z y m e
[ 1 - 7 ] . T h r o m b i n - l i k e
e n z y m e s
[ 8 - 1 0 ]
are
s e r i n e
p r o t e a s e s , w h i c h
b e l o n g
t o
t h e
t r y p s i n
f a m i l y
[ 1 1 ] .
I n
c o n t r a s t w i t h
t h r o m b i n ,
w h i c h
c o n v e r t s
f i b r i n o g e n
t o f i b r i n
b y r e m o v i n g
f i b r i n o p e p t i d e s
A
a n d B , a t h r o m b i n - l i k e
e n z y m e r e l e a s e s
o n l y f i b r i n o p e p t i d e A
f r o m
f i b r i n o g e n
[ 1 2 , 1 3 ] .
P a r e n t e r a l a d m i n i s t r a t i o n
o f
t h e
enzyme
causes t h e
c o n v e r s i o n
o f
f i b r i n
i n t o a
f i b r i n d e r i v a t i v e t h a t
i s
r a p i d l y d e g r a d e d a n d
e l i m i n a t e d
t h r o u g h a f i b r i n o l y t i c
process i n
t h e b l o o d s t r e a m
[ 1 2 ] . A n c r o d
i s
a h i g h l y a c t i v e
t h r o m b i n - l i k e
e n z y m e p u r i f i e d f r o m t h e venom o f
C a l l o s e l a s m a
r h o d o s t o m a
[ 1 3 ] .
S i n c e
a n c r o d
h a s
b e e n
c o m m e r c i a l l y
a v a i l a b l e
f o r many
y e a r s , numer ous
e x p l o r a t o r y s t u d i e s on i t s
c l i n i c a l
a p p l i c a t i o n
h a v e
b e e n
r e p o r t e d [ 1 4 ] . T h e
r e s u l t s
h a v e
i n d i c a t e d
c o n s i d e r a b l e
p o t e n t i a l
v a l u e f o r
t h i s
p r o t e i n
i n
t h e t r e a t m e n t o f
v a r i o u s
t h r o m b o - e m b o l i c
s t a t e s , a n d
i n
t h e
p r e v e n t i o n
o f
t h r o m b o s i s .
H o w e v e r , t h e m o l e c u l a r
c h a r a c t e r i s t i c s o f a n c r o d
h a v e n o t b e e n
d e s c r i b e d .
P r e v i o u s l y ,
we
h a v e c l o n e d a n d
a n a l y s e d
t h e
cDNA
f o r a
p l a t e l e t - a g g r e g a t i o n
i n h i b i t o r
a n d f o r a
h a e m o r r h a g i c
p r o t e i n
t h a t
were
d e r i v e d
f r o m
t h e
v e n o m - g l a n d
cDNA
l i b r a r y
o f C a l l o s e l a s m a
r h o d o s t o m a
[ 1 5 ] . H e r e ,
we
r e p o r t
t h e
c l o n i n g
a n d
n u c l e o t i d e - s e q u e n c e
a n a l y s i s
o f
t h e
cDNA
f o r
a n c r o d ,
a n d
t h e
d e d u c e d
a m i n o - a c i d
sequence.
M A T E R I A L S A N D
M E T H O D S
I s o l a t i o n o f c D N A
c l o n e s
f o r a n c r o d
A
s e t
o f
cDNAs
d e r i v e d f r o m
a
p o l y ( A ) +
RNA
p r e p a r a t i o n
o f
t h e
venom
g l a n d s
o f
C a l l o s e l a s m a
r h o d o s t o m a were
c l o n e d
i n t o
t h e
Uni-ZAPTMXR
v e c t o r
[ 1 6 ]
b y i n s e r t i n g
t h e cDNAs i n t o
f l a v o x o b i n ) .
K e y
a m i n o - a c i d
r e s i d u e s ( H i s 4 3 , A s p 8 8 ,
S e r l 8 2 a n d
A s p 1 7 6 )
t h a t are t h o u g h t t o b e i n v o l v e d
i n
t h e s u b s t r a t e c l e a v a g e
a n d i n
t h e s u b s t r a t e - b i n d i n g
r e a c t i o n a r e c o n s e r v e d .
A n c r o d
c o n t a i n s
1 3 c y s t e i n e r e s i d u e s .
B a s e d
o n a l i g n m e n t w i t h
t h e
a m i n o - a c i d
sequences o f
t r y p s i n
a n d
b a t r o x o b i n , s i x
d i s u l p h i d e
b r i d g e s ca n b e
p r e d i c t e d t o b e
p r e s e n t
i n
t h e a n c r o d
p r o t e i n . T h e
e x i s t e n c e o f a
f r e e
c y s t e i n e , w h i c h
c h a n g e s t h e
common
sequence
s u r r o u n d i n g
t h e
S e r 1 8 2 a c t i v e s i t e
f r o m
G l y - A s p - S e r - G l y - G l y - P r o
t o
C y s - A s p - S e r - G l y - G l y - P r o ,
i s
u n u s u a l
f o r
a s e r i n e
p r o t e a s e .
t h e E c o R I a n d
X h o I s i t e s
o f
t h e
Uni-ZAPTMXR
p h a g e
DNA.
F o r
c o n v e n i e n c e ,
t h e
Uni-ZAPTXR
v e c t o r was e x c i s e d i n
v i v o
t o
g e n e r a t e a p B l u e s c r i p t S K - p h a g e m i d cDNA
l i b r a r y b y co-
t r a n s f e c t i n g
t h e Uni-ZAPMXR v e c t o r a n d
an
R 4 0 8
h e l p e r
p h a g e
[ 1 7 ]
i n t o E s c h e r i c h i a c o l i
X L 1 - B l u e h o s t
c e l l s a c c o r d i n g t o
t h e
m a n u f a c t u r e r s
i n s t r u c t i o n s
( S t r a t a g e n e ,
L a
J o l l a ,
C A , U . S . A . ) .
E . c o l i
X L 1 - B l u e was t h e n
i n f e c t e d
w i t h
t h e
r e s c u e d
p h a g e m i d .
T h e i n f e c t e d
b a c t e r i a
were p l a t e d o u t
on
t o L u r i a
b r o t h / a m p i c i l l i n p l a t e s .
A m p i c i l l i n - r e s i s t a n t
c o l o n i e s
were
t r a n s -
f e r r e d on
t o
n i t r o c e l l u l o s e membrane a n d
p r e p a r e d
f o r
c o l o n y
h y b r i d i z a t i o n
[ 1 8 ] .
A
2 0 m e r i c
o l i g o n u c l e o t i d e
p r o b e
( s e e F i g u r e
1 )
was
s y n t h e s i z e d
a c c o r d i n g
t o
t h e
n u c l e o t i d e
sequence
o f
a
b a t r o x o b i n cDNA
[ 8 ] . T h e
p r o b e
wa s
3 2 P - e n d - l a b e l l e d w i t h T 4
p o l y n u c l e o t i d e k i n a s e
a n d
[ y - 3 2 P ] A T P
[ 1 9 ] .
T h e
m e m b r a n e
r e p l i c a s were
p r e - h y b r i d i z e d
a t 6 5
° C f o r
3
h
i n
h y b r i d i z a t i o n
b u f f e r
[ 0 . 4 5
M
s o d i u m
c h l o r i d e , 0 . 0 4 5
M
s o d i u m
c i t r a t e , 0 . 0 2 M
p o t a s s i u m
p h o s p h a t e , pH 6 . 8 , 0 . 1
( w / v )
S D S ,
0 . 0 2 0
( w / v )
f i c o l l a n d
0 . 0 2 ( w / v )
p o l y v i n y l p y r r o l i d o n e ]
p l u s
100
g / m l
s h e a r e d
a n d
d e n a t u r e d
h e r r i n g - s p e r m
DNA.
T h e
m e m b r a n e
r e p l i c a s
were
t h e n
h y b r i d i z e d a t
5 4 °C
f o r 8 h i n
3 0 m l o f
h y b r i d i z a t i o n
b u f f e r
c o n t a i n i n g
5
p m o l
o f t h e l a b e l l e d
o l i g o -
n u c l e o t i d e
p r o b e .
S u b se qu e nt w a s h es o f t h e
h y b r i d i z e d mem-
b r a n e f i l t e r s were c a r r i e d o u t i n
6
x
SSC
1
x
SSC
i s
0 . 1 5
M
N a C l / 0 . 0 1 5
M s o d i u m
c i t r a t e )
a n d
0 . 1
( w / v ) SDS as
f o l l o w s :
f o u r
t i m e s a t 4
° C
f o r
5 m i n
e a c h ,
once a t room
t e m p e r a t u r e
( 5 m i n ) a n d once a t
5 0 ° C
( 2
m i n ) .
T h e
p o s i t i v e
c l o n e s were
d e t e c t e d
b y
a u t o r a d i o g r a p h y ,
a n d
were f u r t h e r
p u r i f i e d
b y
i s o l a t i n g
s i n g l e
c e l l
c o l o n i e s .
D e t e r m i n a t i o n
o f
t h e n u c l e o t i d e
s e q u e n c e
o f t h e
c D N A i n s e r t
T h e
p h a g e m i d s
o f
p o s i t i v e
c l o n e s
were
p u r i f i e d b y
a l k a l i n e
l y s i s
o f
t h e
h o s t
c e l l ,
f o l l o w e d
b y
e q u i l i b r i u m
c e n t r i f u g a t i o n
i n
a
C s C I / e t h i d i u m - b r o m i d e
g r a d i e n t
[ 1 8 ] .
The n u c l e o t i d e
sequence
o f
t h e
cDNA
i n s e r t was d e t e r m i n e d
b y
t h e
d i d e o x y
method
u s i n g
t h e
w a l k i n g p r i m e r
s t r a t e g y ( F i g u r e
2 ) .
§
T o
whom
c o r r e s p o n d e n c e
s h o u l d b e a d d r e s s e d .
T h e
n u c l e o t i d e
s e q u e n c e
r e p o r t e d
i n
F i g u r e
3
w i l l
a p p e a r
i n t h e GenBank
D a t a b a s e u n d e r
a c c e s s i o n
n o .
L 0 7 3 0 8 .
3 8 7
i o c h e m . J .
1 9 9 3 )
2 9 4 ,
3 8 7 - 3 9 0
P r i n t e d
i n
G r e a t
B r i t a i n
-
8/17/2019 1993 Au Molecular Cloning and Sequence Analysis of the CDNA for Ancrod C. Rhodostoma
2/4
L . - C .
A u a n d o t h e r s
O l i g o n u c l e o t i d e
p r o b e
G AA
CTO
I
G T C
AT T OGA
G G T
G AT
G A k
T O G C
Glu
Lo u
V a l I l e Gly G ly Asp O lu
C ys
Asp
- 1 1
7
F i g u r e
1
O l i g o n u c l e o t i d e
p r o b e ( 2 0 m e r ;
b o x e d )
s y n t h e s i z e d
a c c o r d i n g
t o
t h e n u c l e o t i d e
s e q u e n c e
o f
t h e
N - t e r m i n a l
a m i n o - a c i d
r e s i d u e s
o f
b a t r o x o b i n
[ 8 ]
N h e l
B a l l
P v u l l
B s p M I I
4 0 0
N c o l
P s t I
H p a l
EcoRI
BamHI
P v u l l D r a l
Smal
BamHI
I
8 0 0
2
F i g u r e
2
P a r t i a l
r e s t r i c t o n
m a p
a n d
s e q u e n c i n g
s t r a t e g y
u s e d t o
o b t a i n
t h e
n u c l e o f t d e
s e q u e n c e
o f
t h e
cDNA
c l o n e
J - 5
A r r o w s i n d i c a t e
d i r e c t i o n s a n d
s e q u e n c e d r e g i o n s .
T h e
l o n g e s t
o p e n
r e a d i n g
f r a m e
i s
s h o w n
a s a
f i l l e d
b o x .
RESULTS AND
D I S C U S S I O N
S c r e e n i n g
o f a
cDNA
l i b r a r y
Based o n t h e n u c l e o t i d e
s e q u e n c e
o f
t h e N - t e r m i n a l
a m i n o - a c i d
r e s i d u e s
of
batroxobin
[ 8 ] ,
a
2 0 m e r i c
o l i g o n u c l e o t i d e
was
s y n t h e s i z e d ( s e e F i g u r e 1 )
and wa s u s e d
a s
a
h y b r i d i z a t i o n p r o b e
i n
t h e
s c r e e n i n g
o f
a
p h a g e m i d - b a s e d
cDNA
l i b r a r y
as
d e s c r i b e d
i n
t h e M a t e r i a l s and
metho ds
s e c t i o n . T h r e e
p o s i t i v e
c l o n e s
( d e s i g n a t e d
J - 2 ,
J - 3
and
J - 5 )
were
o b t a i n e d
from
t h e
s c r e e n i n g
o f
about 1000
c o l o n i e s .
T h e s i z e of
t h e
cDNA
i n s e r t
i n
e a c h
p o s i t i v e
c l o n e was
determined
b y
PCR
u s i n g
a
p a i r
o f
p r i m e r s f l a n k i n g
t h e
c l o n i n g
s i t e s
o f
t h e
p B l u e s c r i p t S K - p h a g e m i d
DNA.
The
r e s u l t s
i n d i c a t e d
t h a t
J - 2 ,
J - 3 and
J - 5 were
i n d e p e n d e n t
and
o v e r l a p p i n g
c l o n e s
c a r r y i n g
cDNA
i n s e r t s
o f
1 . 4 ,
1 . 3
and
1 . 5
k b
r e s p e c t i v e l y .
N u c l e o t i d e - s e q u e n c e a n a l y s i s
Clone
J - 5 ,
which
c a r r i e s t h e
l o n g e s t
cDNA
i n s e r t ,
was
c o m p l e t e l y
s e q u e n c e d ( F i g u r e 2 ) .
Most
r e g i o n s
o f
t h e cDNA
were
s e q u e n c e d
i n
both d i r e c t i o n s . On
a n a l y s i s
o f t h e
n u c l e o t i d e
s e q u e n c e
o b t a i n e d ,
a
l o ng o p e n r ea d i n g
frame
wa s
o b s e r v e d
( F i g u r e
2 ) .
I n
t h i s
o p e n r ea d in g f r a m e ,
t h e
t r a n s l a t i o n - i n i t i a t i o n s i t e
i s
a s s i g n e d
t o
t h e f i r s t
methionine
codon
a t
n u c l e o t i d e s
1 7 8 - 1 8 0 ,
which
i s
f o l l o w e d
b y
an
u n i n t e r r u p t e d
c o d i n g
r e g i o n ,
which
p u t a t i v e l y
codes
f o r 258
amino-acid r e s i d u e s
( F i g u r e
3 ) .
The 3
n o n - c o d i n g
r e g i o n
c o n t a i n e d a
p o l y ( A ) + s e q u e n c e
and
a
p u t a t i v e
p o l y a d e n y l a t i o n s i g n a l s e q u e n c e
AATAAA
( n u c l e o t i d e s
1 5 1 9 -
1 5 2 4 ) ,
which
was l o c a t e d a t
a
s i t e
1 4
n u c l e o t i d e s
u p s t r e a m
from
t h e
p o l y ( A ) + s e q u e n c e ( F i g u r e 3 ) .
Two
p u t a t i v e g l y c o s y l a t i o n
s i t e s ,
A s n - X a a - T h r ,
were l o c a t e d a t
amino-acid
r e s i d u e s 1 4 8 - 1 5 0
and 229-231.
Ancrod
i s
known t o
b e a
g l y c o p r o t e i n
[ 2 0 ] .
A m i n o - a c i d
s e q u e n c e
a n a l y s i s
T h e
amino-acid
s e q u e n c e
deduced from
t h e
c o d i n g
r e g i o n
o f
t h e
1
3 1
T G A
C TC T OC C AG
C GT T TG AGA
TT G
TT G
GC C
AC C
C AG C OG
C T T
AAT
T TG
ATC
AAA
TAA
AG T
6 1
9 1
GC T
GC T T WA
TCA
AG A AG T
CT C CTC
T? O
AOTT T
CTG
AT T
AGA
TT G
T Coo
T T
CTC
MG
1 2 1
1 5 1
G 0 T
AAG TT S G 0 G AC T
G 0 0
AT C TT A
C AG O C A
M
AMC
TrCTOr
TO C M G A 0T
GA A
G C T AT G
1 8 1 2 1 1
4
G T G C T G
AT C
AG A GT G
C T A
GC A
AAC
C TT G TG
ATA C T A
CM
CT ? TCT
T AC
GC A
CAA AA G
TC T
va l
l e u
ile
arg
va l
l e u
ala
a n l eu v a l
il e l eu gl n le u
m er
tyr
al a
gl n
ly e
se r
2 4 1 2 7 1
- 4
T C T G AA
CTG IGTC
Arr
GC A
OG T G AT G AA TG T AAC AT A AA T GA A
CAT
C G T
T T C
C TT G TA GC C
ser
gl u
le u
va l
il e
gl y
g l y
asp
gl u
cys
as n
i l e
aen gl u h is
ar g
ph e
le u
val al a
3 0 1 - 1 1 3 3 1 1 7
TT G TA T G AC
AG T
AC G
AC T
C GG AAT
m T T C TC
TG T G GT G GG
G TT ITO AT C C AT
C C G GA A
TG G
lo u ty r
as p
se r
t hr t hr
arg
as n ph e
l e u
cys
gl y gl y
va l
l e u il e h is
pro gl u
tr p
3 6 1 3 9 1 3 7
G T G
AT C AC T
O CT
AAA
C XC TG C
AAC
AAG
AM AGT AT G
G TC C TA
TA C
C TT
G O T AAG C AT
AAA
v al
i l e
th r al a ly s his
cy s
aen
l ye l ye
me r
m e t
va l le u t y r
l e u gl y
l y s his
1 y m
4 2 1 4 5 1
9 7
C AA AG T G TA AM
T r r
G AC
GA T G AG C AG
G M A AG A T TC
C C A AAG
G AG A AG C AC
TT T
AT T
C GC
gl n m er
va l
ly s
ph e a sp a sp gl u
gl n
gl u ar g ph e
pro
ly e gl u ly e hi s ph e ile
ar g
4 8 1
*77
T G T AAC AAA CC C C GT ACC
AG A
TG G
GG C
G AG G AC
AT C AT G
T T G
ATC
AG
CT
AAC AAA
C CT
cy m as n
l y e
pro ar g
th r ar g
t rp gl y gl u as p ile
m e t l e u
i l e
ar g le u asn ly s pro
5 4 1 5 7 1 9 7
G T T AAC AAC AG T
G AA
C AC AT C
GC T
C CT C T C AMC T T I C C T TC C AAC C C T C C C AT T GT G GG C
va l as n ao n
m e r
gl u h is i l e al a pro le u ser
le u
pro se r as n p ro p ro i l e va l
g l y
6 0 1
6 3 1 1 1 7
T C A
G TT
TG C
CG T G TT ATG
GG A TG G GO C TC A AT C AAT
AAA
T AT AT A
GA C
G T T
TT O C C C
GAT
me r
v al cy s ar g v al m et gl y
t rp g ly
m e r il e ag n l y s
ty r
i l e
asp
va l l e u pro
am
6 6 1
6 9 1
1 3 7
G AA C C T
C GT T GT
GC T AAT
A?
AAC
C TG
T AC
AAT TAC
AC G
G TO
TG T
C O T
GG A GT T
TT
C C A
gl u
pro
arg
cy s
al a as n
ile
0sn
le u
ty r
asn
ty r th r
va l
cy s
arg
gl y
vo l
ph
pro
7 2 1
7 5 1 1 5 7
AG G AT A C C A AA G A MA
AG C
AAA AT A TTG TG T G CA
GG T G AC C TG
C AA O GA CGC C TA GAT
TC A
ar g i l e
pro ly s l y s
se r
ly e
i l e l e u
cy s al a gl y a sp le u
gl n
gl y ar g
l e u
asp
m e r
7 8 1
8 1 1
1 7 7
TO T
C AC
TG T
G AC T CT
G G G
GO A
C C T
C TC AT T
T GT AG T
G AA G AA TT C
C AT
GOC AT T
G TA T AT
cy s h is cy s as p
se r
gl y gl y pro le u i l e
cy s smr
gl u gl u ph e hi s gl y ile va l
ty r
8 4 1
8 7 1 1 9 7
C G G
G GA C CC AAT C C T
T G T 0C C
C AA
C C A GA T AAG C C T
GC C C T C TAC AC C AAC ATC TTC GA T
ar g
gl y
pro a n pro
cy s al a gl n pr o
a sp l ys
pro al a
l a u
ty r
th r
as n i l e
ph e
asp
9 0 1
9 3 1 2 1 7
C AT C TT
C AC
TO G
AT C
C TT
AG C
AT T
AT G G C A
GG A
AAT GC A AC T TG C T AT C C A T AA
AAC C T T
hi s le u
hi s t rp
i l e l eu me r
il e m et al a glylaon
o l a
thrcys
ty r
pr o
9 6 1
9 9 1 Z 3 4
TT G
A AA T AG TT A
AG T
O G A GM
AAT G TA AC A
T A T
T AG T AA AT C
TC T
T C T
ATA
TC C ST G C AT
1 0 2 1
1 0 5 1
T G G AAC
AT A
TT C
C C A GC T
GT A AG C
C? ? T?? AG A
C TC
AM TAG
G AC
TA C C ? T T O G G
MT
AAG
1 0 8 1 1 1 1 1
M A G TG C
T C A AM
TA G
TG C TO C A GG G AT
C AT
??C
C C A
P T T
AAT
TT C
AG T ?? A
AM
C AG
TO
1 1 4 1
1 1 7 1
CC
TAG
AT T G G A
G O A
C TG T AG
G O T
TAG
G T
CAA AAT T?? CT G
AC T
C T A
AM TO G A C C
1 2 0 1
1 2 3 1
A ? ?
C C A
AT C ?? T TAA
C C T C TG
AAT
A
TTC
AAT TTC ? T C AG C? C TG
G 0 C AG AT C T O C
1 2 6 1
1 2 9 1
TC T
G A O C ? ?
GT C ST C
T G O
C AG
AC A T TT C AT
TA C C C A
OC T A G O TA A
C AT
C AT CM TO C
TAG
1 3 2 1
1 3 5 1
AAT A?T ? ? T ??C TA T TG O TA T ?? C T O T G G C
A? ?
TA C AAT AC G C TC
? T A
T O MT T AT C CA
1 3 8 1 1 4 1 1
G TC AC C
C C A
CA M
AC A T AT
AC A
T AT
C C C
CO G GT C C C A C T T T G
C C T A M A AM G A T C C C
A G A
1 4 4 1
1 4 7 1
? ? A
AC C
CCC
CAT
TT C
CCA
AT C AC T
A AA T AG
AAT
C T T I T I
AG A
AT O
AT G
TT T TC A WT T AAA
1 5 0 1 1 5 3 1
TT C
T TA
G G T
AT C
C AC AAC
AAT AAA
T TG
T AT
AAA T CA T CA
AM AM
AM
AM AM
M A M A
F i g u r e
3
N u c l e o t i d e
s e q u e n c e
o f
t h e a n c r o d c O N A
J - 5
a n d t h e p r e d i c t e d
a m i n o - a c i d
s e q u e n c e
o f
t h e a n c r o d
p r e c u r s o r
p r o t e i n
T h e
n u c l e o t i d e
r e s i d u e s
a r e
n u m b e r e d
o n
t h e
l e f t
a n d
t h e
a m i n o - a c i d
r e s i d u e s
a r e
n u m b e r e d
o n
t h e
r i g h t .
T h e
a m i n o - a c i d
n u m b e r i n g
s t a r t s a t t h e
N - t e r m i n a l
a m i n o a c i d , v a l i n e o f a n c r o d .
T h e
p u t a t i v e p r e - p e p t i d e a n d
p r o - p e p t i d e c o n s i s t o f a m i n o a c i d r e s i d u e s
- 2 4
t o -7 a n d -6
t o
-1
r e s p e c t i v e l y .
A r r o w s
i n d i c a t e
p r e d i c t e d
c l e a v a g e
s i t e s . A s t e r i s k s i n d i c a t e
p u t a t i v e a m i n o -
a c i d
r e s i d u e s t h a t
a r e i n v o l v e d
i n
c a t a l y t i c
a c t i o n . T h e
p u t a t i v e g l y c o s y l a t i o n
s i t e s
ar e s h o w n
i n
b o x e s
a n d
t h e
p o l y a d e n y l a t i o n - s i g n a l s e q u e n c e ,
A A T A A A , i s
u n d e r l i n e d .
J - 5
cDNA
( F i g u r e 3 )
was
c o m p a r e d w i t h t h a t o f t h e b a t r o x o b i n
precursor ( s e e
F i g u r e 4 )
[ 8 ] . B a t r o x o b i n i s
a t h r o m b i n - l i k e
e n z y m e
t h a t i s
d e r i v e d f r o m
s n a k e
venom o f B o t h r o p s atrox
m o o j e n i .
O n
a l i g n m e n t
u s i n g t h e m e t h o d
o f
P e a r s o n a n d Lipman
[ 2 1 ] ,
60
sequence
i d e n t i t y
( 7 0 0
s i m i l a r i t y
c o n s i d e r i n g
con-
s e r v a t i v e
s u b s t i t u t i o n s )
was
found b e t w e e n
t h e
J - 5 - d e r i v e d
a m i n o -
a c i d
s e q u e n c e
and
t h a t o f
b a t r o x o b i n
[ 8 ] .
T h i s
i m p l i e s
t h a t t h e
3 8 8
..
I
I
a
m
m
-
8/17/2019 1993 Au Molecular Cloning and Sequence Analysis of the CDNA for Ancrod C. Rhodostoma
3/4
M o l e c u l a r
c l o n i n g
a n d
s e q u e n c e a n a l y s i s
o f c D N A f o r a n c r o d
t i d e
- p e p t i d e
- 2 4 - 7
1
XV
L
I R
V L A
N
L
V
I
L
Q
L S Y A
Q
8
L
X
V
L I R
V L A N L L
I
L
Q V 8
Y
A
Q
K
8
8
Z
L
2 0
V
I
G
D 8
C N I
N
b
N
R
P L V A
L
Y D
ST
T
R
N
r
L C
Q
a
V I
GG
C D
I
N 5
NF
A Y y
8 P R
YT
C
GX
V I
G
C
N I N
H
F L V L
Y
DA
W
8 G R
F
L
C
G
I G
Q
A
B
a
L S
P
W
Q
VN
r
R
8 P
Q
L
L
C
a
A
I
V
G
G
Y
T
C
G A
N
T
V P Y
Q
V 8
L
Q
a
Q
Y
H
C
V
a
TSS
W
2 P
O V
8
L
Q
V K
L
T
Q
R H
L
C
a
4 0
-
V L I
P
W
T
T A
R
C
N
xK V
L
T
L I
N
Q
V L T A
H
C
N
R R
T L I PB
LT
H DS
K
F
S
L
I
S D RW
L
T A
H L L
Y
P
P
WK8F
T
V
D
D
L
L
V
S
L
I N
8 Q
VB
H
Y
8 a
G V
S
L IG
Q
W
L T
A A
H
C
F
D
G
LPL
Q
D
V
6 0
Y
L G
KN
K
Q
S
K
D D 2
Q
Z
F P
K 5
K
N
I R C N
R
I
H L
G
K H A
G
S
A N
T V
V
R Y P K K F I CN
K
L
G
NH
Q
K
L N D
O
I
R
N
P
a r r C N
K
R
I G
K
S T R Y
ZK
8
K
S
X
L
D K
I Y I
H
R
Y
R L
G Q N
I
N
V
V
Z
N
Q Q
FI
A
SKS
I
V
H
P
8
Y
R I
Y S a
I
N L
S
I
TK
T P
r
s
QIKIII Q
N
8 0
1 0 0
K
R T R
NG
D I
N
I R
L
N
K
V
N
N
S8 I
N I
A
P L
S
P
KK
N
V
I
T
D K D
I
L
I
R
L
D R
P V
K N 8
N
P
L L
P
X
N T
R
L D
K
I
N
I
R
D
S
V
S
8
S
H I A
P L
S P
N W K
e
L D R D
I
A
L
L
KK
P
I
LS
Y I
H P V
C L P
N
S N T
L
N
N
D I
N
I
K
X
S
A
S
N 8
R V
A
I
8
L
P
Y
K V
8
S
a
N
H
D
I
A L
I
R
Q
A P
L N
Y
T
F Q
K
I
L P
12 0
S
N
P P I V
G
S
V
C
R
VK
W
G
S
N K Y I D V
S N
P
P
S VS
C
R I G
W
A
I T T
S
D
T
SSPPS
V
S
V
C
R INW
S
I T P V T
D R
T A AK
L
H
G
F K
G
R V
T
a
W
a
N
R
R
3T T
S
V
T
S C
A
S
A
G
T
Q
C
L
I
S
G
W
N
T K
S S G
T
S
S
K
D
T
S T
I
Y
T N
C
N
T
GNG
S
K
O
B
I
1 40
1 60
L P D P
R
C
A
N
I
N L Y
N Y
T V
C R
G
Z
P R I
P
K
K
Y
P
D V
P
H
C A N I
N
L
P N N T
V
C R
Y N
G
P
F
P D
V
P H C A N I N
L
L D D V
C
K
P
a
Y P
L
L P 8
A
3
Q
P
S
V LQV V N
L
P L V
R
P
C
K
A
ST
I R
I T
Y
P D V
L
K
C L
K
P I L
S
S
S
K
S A Y
P
a o
I
T S
Q
I
L
Q
K
V
N
I P L
T
N
R
8
C
Q
K
R Y
O
D
Y
K I
T
1 8 0 .
S K I L
C A
G
L
Q
G
L D
S C H C
D
S a
G
P
L
I
C
A K T L C
a
v
L
Q
a G
I
T
C
G
D
SGG
P
L
I
C
Y R T L
C
A
G V
L
Q
G
c
I D
T C
G F D
S
T P
L
I
C
N D
F C
A
G
Y
K
P G G
K
G
D
C
C
G
SG
7
F
V
K
S
N F C A
G
Y L
G G K D
S C
Q
G
S G G P
V
V C
Q
V
C
A
G
K
G G K
D
A
C K G D
S
G
Q
P
L
V C
2 0 0
S E1 Z H
G
V
Y
RG
N P C A
Q P D
K
A L
Y
T N I
N
G
Q
FQ
G I L S
I
G S
D P C A R
P
R
K
A F Y
T
K
V
N G
Q
FQ
G I
V
Y
I G
8 H P C G
Q
8 R K P
G I Y T
K
P Y N
N
R
W Y
Q
N
I V S W
G G
C
D R N
GK
G
F Y T H V
S
G K L
Q
G I V
S
G S
G
A Q
KK
G
V
Y T
R
V
K H N G
N
R L
V G I T S G
2
G C
A
R R
Q
P
G V
Y T V
2 2 0
F
D
H
L HNI L
S
I
N
G
N
A
T
Y P
F
Y
L P
Q
S
I A
GKA
C
P
D
Y
N A
W
I
Q
S
I A G N
T
A
T
C L P
F
R L
K K W I K
V
I
D
R
L G
S
C
N
Y
V
S
W I
K
Q
T
A S N
Y
N
W
I L
B
K
T
S
8
D
G
A Q
KQS
8
P
A
a n c r o d i s v a l i n e
[ 2 0 ] . T h e h y d r o p h i l i c p e p t i d e ( a m i n o
a c i d s
-6
t o - 1 )
i s p r e s u m a b l y a z y m o g e n p e p t i d e .
F i g u r e 4
a l s o s h o w s a h i g h s e q u e n c e s i m i l a r i t y b e t w e e n
a n c r o d
a n d o t h e r m a t u r e s e r i n e
p r o t e a s e s [ 9 , 2 2 , 2 3 ] .
B a s e d
o n
s e q u e n c e
s i m i l a r i t y , w e c o u l d d e d u c e
t h e c a t a l y t i c
a m i n o - a c i d r e s i d u e s
t o
b e
H i s 4 3 , A s p 8 8 a n d S e r 8 1 2 ( F i g u r e
4 ) .
T h e l o c a t i o n
o f
A s p 1 7 6 a t
a p o s i t i o n e q u i v a l e n t
t o
t h e b o t t o m o f t h e
s u b s t r a t e - b i n d i n g
p o c k e t i s
c o n s i s t e n t
w i t h
t h e
f a c t
t h a t
a n c r o d
c l e a v e s
a
f i b r i n o p e p t i d e
A f r a g m e n t
f r o m
f i b r i n o g e n
a f t e r
t h e
p o s i t i v e l y
c h a r g e d a r g i n i n e r e s i d u e
[ 1 3 ] .
A n c r o d
c o n t a i n s 1 3
c y s t e i n e
r e s i d u e s .
B a s e d o n
c o m p a r i s o n
o f
t h e a l i g n e d s e q u e n c e s o f
b a t r o x o b i n ,
f l a v o x o b i n
a n d
t r y p s i n ,
s i x
p o s s i b l e
d i s u l p h i d e b r i d g e s
a p p e a r
t o
b e
C y s 7 _ C y s I 4 1 ,
C y s 2 8 - C y s 4 4 ,
C y s 7 8 _ C y s 2 3 2 , C y s 1 2 0 _ C y s 1 8 8 , C y s 1 5 2 _ C y s t 6 7
a n d
C y s 7
- C y s 2 0 3
T h e
e x i s t e n c e
o f a f r e e
c y s t e i n e
( C y s 1 8 0 ) ,
w h i c h
c h a n g e s
t h e
common
s e q u e n c e
s u r r o u n d i n g
t h e
a c t i v e
s i t e
S e r 1 8 2 f r o m
G l y -
A s p - S e r - G l y - G l y - P r o t o
C y s - A s p - S e r - G l y - G l y - P r o ,
i s
u n u s u a l
f o r
a
s e r i n e
p r o t e a s e .
To e n s u r e t h a t a m i n o - a c i d r e s i d u e s
u n i q u e
t o a n c r o d
( s u c h
a s
C y s 1 8 0 , L y s 4 2 , A r g t 7 4 )
f o u n d
i n
t h e
s e q u e n c e
a l i g n m e n t ( F i g u r e 4 ) w e r e n o t a r t e f a c t s
d e r i v e d
f r o m t h e cDNA
c l o n i n g
p r o c e s s ,
a n
i n d e p e n d e n t c l o n e
J - 2
w a s a l s o
s e q u e n c e d .
T h e
s e q u e n c e
o f
J - 2
w a s
c o n s i s t e n t
w i t h
t h a t
o f
t h e
J - 5 c l o n e .
T h e r e p l a c e m e n t o f
G l y 8 0
b y
a
c y s t e i n e r e s i d u e
i n t h e common
s e q u e n c e a r o u n d t h e s e r i n e r e s i d u e
o f
t h e a c t i v e s i t e d o e s n o t
s e e m t o d e c r e a s e t h e t h r o m b i n - l i k e
p r o t e o l y t i c
a c t i v i t y
o f a n c r o d
c o m p a r e d
w i t h t h a t o f b a t r o x o b i n
[ 8 ] .
F i n a l l y ,
w e
s p e c u l a t e
t h a t a n c r o d
may
a l s o
p o s s e s s
a
c y s t e i n e
p r o t e a s e - l i k e
a c t i v i t y .
C y s t e i n e p r o t e a s e s
s h a r e
s o m e
s i m i l a r i t i e s
o f r e a c t i o n m e c h a n i s m
w i t h
s e r i n e
p r o t e a s e s
[ 2 6 ] ,
b u t
t h e y
a r e
g e n e r a l l y
a c t i v e i n
a h i g h e r
pH
r a n g e .
T h e - S H
g r o u p
o f a f r e e
c y s t e i n e
r e s i d u e
p l a y s
a
r o l e
t h a t
i s s i m i l a r
t o
t h a t o f t h e
-OH
g r o u p
o f t h e a c t i v e - s i t e
s e r i n e .
C y s 1 8 0 ,
w h i c h
i s
l o c a t e d
c l o s e t o
t h e
c a t a l y t i c c a v e ,
may
b e
c o u p l e d
w i t h
H i s 4 3 ,
a s
g e n e r a l l y
o c c u r s
i n
c y s t e i n e
p r o t e a s e s
t o
c l e a v e p e p t i d e - b o n d s .
W e
t h a n k
D r .
M i n g - Y i
L i a u
f o r
p r o v i d i n g
u s
w i t h
v e n o m
g l a n d s . W e a l s o
t h a n k
D r .
T u r - F u
H u a n g
f o r
h e l p f u l a d v i c e a n d
D r .
K o n g - B u n g C h o o
f o r
c r i t i c a l
r e a d i n g
o f
t h e
m a n u s c r i p t .
T h i s
w o r k
w a s
s u p p o r t e d
b y g r a n t
N S C 8 2 - 0 4 1 8 - B 0 7 5 - 0 1 6 - B C
f r o m
t h e
N a t i o n a l
S c i e n c e
C o u n c i l
o f t h e
R e p u b l i c
o f C h i n a .
F i g u r e
4
A l i g n e d a m i n o - a c i d
s e q u e n c e s
o f
t h e
a n c r o d p r e c u r s o r ,
b a t r o x o b i n
p r e c u r s o r ,
f l a v o x o b i n ,
b o v i n e
t h r o m b i n ,
b o v i n e
t r y p s i n a n d h u m a n
k a l l i k r e i n
REFERENCES
T h e
n u m b e r i n g
s t a r t s
f r o m
t h e N - t e r m i n a l r e s i d u e
o f a n c r o d . G a p s
h a v e
b e e n i n t r o d u c e d
t o
o p t i m i z e
t h e
s e q u e n c e
s i m i l a r i t y .
A s t e r i s k s
i n d i c a t e t h e a m i n o - a c i d
r e s i d u e s t h a t a r e
i n v o l v e d
i n t h e
c a t a l y t i c
a c t i o n .
cDNA c l o n e
J - 5
d o e s
e n c o d e
f o r
a n c r o d ,
w h i c h
i s a l s o
a
t h r o m b i n -
l i k e s e r i n e
p r o t e a s e . M o r e o v e r , a 9 3
sequence
i d e n t i t y
b e t w e e n
t h e
f i r s t 3 0 a m i n o - a c i d
r e s i d u e s
o f
t h e
N - t e r m i n i h a s a l l o w e d us
t o
p r e d i c t
t h a t a n c r o d
i s a l s o
s y n t h e s i z e d
as
a
pre-zymogen
b e f o r e
m a t u r a t i o n ,
as
i s
b a t r o x o b i n
a n d
o t h e r s e c r e t o r y s e r i n e
p r o t e a s e s [ 8 , 2 2 - 2 4 ] .
T h e
precursor p e p t i d e i n c l u d e s
a
s i g n a l
p e p t i d e ( p r e - p e p t i d e )
a n d a
z y m o g e n
p e p t i d e
( p r o - p e p t i d e ) .
I t i s
known
t h a t
s i g n a l p e p t i d e s
g e n e r a l l y
c o n t a i n
a
r e g i o n
t h a t i s r i c h
i n
h y d r o p h o b i c a m i n o - a c i d
r e s i d u e s
w i t h
l a r g e
s i d e - c h a i n s ,
a n d
t e r m i n a t e
i n
a
r e s i d u e
h a v i n g
a
s m a l l
s i d e - c h a i n
( A l a ,
S e r ,
or
G l y )
[ 2 5 ] . T h e r e f o r e ,
we
p r e d i c t
t h a t
a
p o s s i b l e
s i t e f o r
t h e
c l e a v a g e
o f
t h e
s i g n a l p e p t i d e
i n t h e
precursor
i s
p r e s e n t
a f t e r t h e A l a r e s i d u e
a t
p o s i t i o n
-7
( t h a t
i s ,
t h e
s i g n a l
p e p t i d e
covers t h e
sequence
f r o m
a m i n o
a c i d s
-2 4 t o
- 7 ; F i g u r e
3 ) .
The
z y m o g e n p e p t i d e
i s
known
t o c o n t a i n
a
r e g i o n
t h a t i s
r i c h
i n
h y d r o p h i l i c
a m i n o -
a c i d r e s i d u e s
[ 8 , 2 2 , 2 3 ]
a n d t h e
f i r s t
N - t e r m i n a l a m i n o a c i d o f
1
O u y a n g , C . , T e n g ,
C . M . a n d
H u a n g ,
T . F .
( 1 9 8 7 )
A s i a P a c .
J . P h a r m a c o l .
2 ,
1 6 9 - 1 7 9
2
E s n o u f ,
M . P .
a n d
T u n n a h ,
G . W .
( 1 9 6 7 )
B r . J .
H a e m a t o l .
1 3 , 5 8 1 - 5 9 0
3
P i z z o ,
S .
V . ,
S c h w a r t z ,
M .
L . ,
H i l l ,
R .
L . a n d
M c k e e ,
P .
( 1 9 7 2 )
J . B i o l . C h e m .
2 4 7 ,
6 3 7 - 6 4 5
4
O u y a n g , C . ,
H w a n g , L .
J . a n d
H u a n g ,
T .
F .
( 1 9 8 3 )
T o x i c o n
2 1 ,
2 5 - 3 3
5
O u y a n g , C . ,
H w a n g , L .
J .
a n d
H u a n g ,
T .
F .
( 1 9 8 5 )
T a i - W a n
H s u c h
H u i
T s a
C h i h
84 ,
1 1 9 6 - 1 2 0 6
6
O u y a n g ,
C . , Y e h ,
H .
a n d
H u a n g ,
T .
F .
( 1 9 8 6 )
T o x i c o n
2 4 ,
6 3 3 - 6 4 3
7
H u a n g ,
T .
F . ,
W u ,
Y .
I.
a n d
O u y a n g ,
C .
( 1 9 8 7 )
B i o c h i m .
B i o p h y s . A c t a
9 2 5 ,
2 4 8 - 2 5 7
8
I t o h , N . , T a n a k a , N . , M i h a s h i ,
S . a n d Y a m a s h i n a I .
( 1 9 8 7 )
J .
B i o l .
C h e m .
2 6 2 ,
3 1 3 2 - 3 1 3 5
9
S h i e h ,
T .
C . , K a w a b a t a ,
S .
I
K i h a r a , K . , O h n o ,
M .
a n d
I w a n a g a , S .
( 1 9 8 8 )
J . B i o c h e m .
( T o k y o ) 1 0 3 ,
5 9 6 - 6 0 5
1 0
P i r k l e ,
H . , T h e o d o r ,
a n d
L o p e z ,
R .
( 1 9 8 9 )
T h r o m b . R e s .
5 6 ,
1 5 9 - 1 6 8
1 1
I t o h , N . , T a n a k a , N . ,
F u n a k o s h i , I K a w a s a k i ,
T . ,
M i h a s h i ,
S . a n d
Y a m a s h i n a ,
I.
( 1 9 8 8 )
J . B i o l .
C h e m .
2 6 3 ,
7 6 2 8 - 7 6 3 1
1 2
S t o c k e r ,
K .
( 1 9 7 8 )
H a n d b . E x p . P h a r m a c o l . 4 6 , 4 5 1 - 4 8 4
1 3
E w a r t ,
M .
R . ,
H a t t o n ,
M . W .
C . , B a s f o r d ,
J . M .
a n d
D o d g s o n ,
K .
S .
( 1 9 7 0 )
B i o c h e m .
J .
1 1 8 ,
6 0 3 - 6 0 9
1 4
L a t a l l o ,
Z .
S .
( 1 9 8 3 )
T h r o m b .
H a e m o s t a s i s
5 0 ,
6 0 4 - 6 0 9
1 5
A u ,
L .
C . , H u a n g ,
Y .
B . ,
H u a n g ,
T .
F . ,
T e h ,
G .
W . , L i n ,
H . H . a n d
C h o o ,
K .
B .
( 1 9 9 1 )
B i o c h e m .
B i o p h y s .
R e s .
C o m m u n .
1 8 1 ,
5 8 5 - 5 9 3
1 6 G r e e n e r ,
A .
( 1 9 9 0 ) S t r a t e g i e s
i n
M o l e c u l a r
B i o l o g y 3 ,
5 - 6
1 7
R u s s e l , M . , K i d d ,
S .
a n d
K e l l y ,
M .
R .
( 1 9 8 6 )
G e n e
4 5 ,
3 3 3 - 3 3 8
3 8 9
A n c r o d
p r e - p r o - p e p
B a t r o x o b i n
p r e - p r o -
A n c r o d
B a t r o x o b i n
F l a v o x o b i n
B o v i n e t h r o m b i n
B o v i n e
t r y p s i n
Human k a l l i k r e i n
A n c r o d
B a t r o x o b i n
F l a v o x o b i n
B o v i n e t h r o m b i n
B o v i n e
t r y p s i n
Human
k a l l i k r e i n
A n c r o d
B a t r o x o b i n
F l a v o x o b i n
B o v i n e t h r o m b i n
B o v i n e
t r y p s i n
Human k a l l i k r e i n
A n c r o d
B a t r o x o b i n
F l a v o x o b i n
B o v i ne t h r o m b i n
B o v i n e
t r y p s i n
Human
k a l l i k r e i n
A n c r o d
B a t r o x o b i n
F l a v o x o b i n
B o v i ne t h r o m b i n
B o v i n e
t r y p s i n
Human
k a l l i k r e i n
A n c r o d
B a t r o x o b i n
F l a v o x o b i n
B o v i ne t h r o m b i n
B o v i n e
t r y p s i n
Human
k a l l i k r e i n
A n c r o d
B a t r o x o b i n
F l a v o x o b i n
B o v i ne t h r o m b i n
B o v i n e
t r y p s i n
Human k a l l i k r e i n
A n c r o d
B a t r o x o b i n
F l a v o x o b i n
B o v i ne t h r o m b i n
B o v i n e
t r y p s i n
Human
k a l l i k r e i n
A n c r o d
B a t r o x o b i n
F l a v o x o b i n
B o v i n e t h r o m b i n
B o v i n e
t r y p s i n
Human k a l l i k r e i n
-
8/17/2019 1993 Au Molecular Cloning and Sequence Analysis of the CDNA for Ancrod C. Rhodostoma
4/4
3 9 0
L . - C .
A u
a n d o t h e r s
1 8
S a m b r o o k ,
J . ,
F r i t s c h , E .
F . , M a n i a t i s ,
T .
( 1 9 8 9 )
i n
M o l e c u l a r C l o n i n g ,
A
L a b o r a t o r y
M a n u a l ,
2 n d
e d n . , c h a p t e r 1 , C o l d S p r i n g
H a r b o r
L a b o r a t o r y ,
C o l d S p r i n g H a r b o r ,
N e w
Y o r k
1 9
M a x a m ,
A .
M . a n d G i l b e r t ,
W .
( 1 9 8 0 ) M e t h o d s E n z y m o l . 6 5 , 5 0 7 - 5 2 1
2 0
H a t t o n ,
M . W . C .
( 1 9 7 3 )
B i o c h e m . J .
1 3 1 , 7 9 9 - 8 0 7
2 1
P e a r s o n ,
W .
R . a n d
L i p m a n ,
D .
J .
( 1 9 8 8 )
P r o c . N a t l .
A c a d . S c i . U . S . A . 8 5 ,
2 4 4 4 - 2 4 4 8
2 2
M a c D o n a l d , R .
J . , S t a r y ,
S . J .
a n d
S w i f t ,
G . H .
( 1 9 8 2 ) J . B i o l .
C h e m .
2 5 7 ,
9 7 2 4 - 9 7 3 2
2 3 S w i f t , G . H . ,
D a r g o n , J .
C . ,
A s h l e y ,
P .
L . ,
S c o t t ,
W . , C u m m i n g s ,
S .
W .
a n d
M a c D o n a l d ,
R .
J .
( 1 9 8 2 )
P r o c .
N a t l .
A c a d . S c i . U . S . A .
7 9 ,
7 2 6 3 - 7 2 6 7
2 4
D e g e n ,
S .
J .
F . , M a c G i l l i v a r y ,
R .
T .
A . a n d
D a v i e ,
E . W .
( 1 9 8 3 )
B i o c h e m i s t r y
2 2 ,
2 0 8 7 - 2 0 9 7
2 5
S t e i n e r ,
D .
F . , Q u i n n ,
P .
S . ,
C h a n ,
S .
J . ,
M a r s h , J .
a n d
T a g e r ,
H .
( 1 9 8 0 ) A n n .
N . Y .
A c a d . S c i .
3 4 3 , 1 - 1 6
2 6 T a k i o ,
K . ,
T o w a t a r i ,
T . , K a t u n u m a , N . , T e l l e r ,
D . C .
a n d T i t a n i ,
K .
( 1 9 8 3 )
P r o c .
N a t l .
A c a d .
S c i .
U . S . A .
8 0 ,
3 6 6 6 - 3 6 7 0
R e c e i v e d 2 1 J a n u a r y 1 9 9 3 / 1 5 M a r c h 1 9 9 3 ; a c c e p t e d 2 2
M a r c h
1 9 9 3