volume 5 Number 7 July 1978
Nucleic Acids Research
Nucleotide sequence at the 5' end of ovalbumin messenger RNA from chicken
Daniel Kuebbing and Charles D.Liarakos
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA
Received 17 May 1978
ABSTRACT
DNA s e q u e n c e a n a l y s i s o f 3 0 0 n u c l e o t i d e s f r o m t h e r e g i o n o f
c l o n e d , d o u b l e - s t r a n d e d o v a l b u m i n c D N A c o r r e s p o n d i n g to t h e 5 ' e n d
of o v a l b u m i n m e s s e n g e r RNA w a s a c c o m p l i s h e d u s i n g t h e t e c h n i q u e o f
Maxam and Gilbert (Proc. Nat. Acad. Sci. U S A (1977) 7 4 . 5 6 0 - 5 6 4 ) .
T h e A U G i n i t i a t i o n c o d o n w a s l o c a t e d 5 2 n u c l e o t i d e s f r o m the A T
l i n k e r s u s e d in c l o n i n g a n d i m m e d i a t e l y a d j a c e n t to t h e a m i n o
t e r m i n a l p e p t i d e o f o v a l b u m i n , i n d i c a t i n g the a b s e n c e o f a " s i g n a l
p e p t i d e " in this p r o t e i n . T h e n u c l e o t i d e s e q u e n c e c o d i n g f o r a
phosphorylated peptide from ovalbumin was also found.
These
results d e m o n s t r a t e that t h e coding portion of m R N A o v b e g i n s near
the 5' e n d o f the m o l e c u l e l e a v i n g s o m e 6 0 0 n u c l e o t i d e s o f n o n coding information a t the 3' e n d .
INTRODUCTION
T h e s u c c e s s f u l c l o n i n g o f the d o u b l e - s t r a n d e d D N A c o p y o f
o v a l b u m i n m e s s e n g e r R N A in p l a s m i d p O V ^ n n (1) h a s p r o d u c e d e n o u g h
D N A to p e r f o r m DNA s e q u e n c e a n a l y s i s b y t h e c h e m i c a l d e g r a d a t i o n
p r o c e d u r e o f M a x a m a n d G i l b e r t ( 2 ) . U s i n g this t e c h n i q u e , w e
have determined the nucleotide sequence of a contiguous series o f
r e s t r i c t i o n e n d o n u c l e a s e f r a g m e n t s f r o m the r e g i o n o f o v a l b u m i n
c D N A d s c o r r e s p o n d i n g to t h e 5 1 e n d o f o v a l b u m i n m R N A ( 3 ) . D N A
s e q u e n c e a n a l y s i s o f t h i s r e g i o n w a s c o n s i d e r e d n e c e s s a r y to u n a m b i g u o u s l y d e t e r m i n e , ( a ) t h e l o c a t i o n o f b o t h the A U G i n i t i a t i o n
codon
a n d the 1161 n u c l e o t i d e c o d i n g p o r t i o n o f m R N A
within the
1 8 9 0 n u c l e o t i d e m e s s e n g e r RNA m o l e c u l e ; a n d (b) the p r e s e n c e o r
absence of a nucleotide s e q u e n c e coding for a "signal p e p t i d e " .
T h e s i z e and s e q u e n c e o f t h e 5 ' n o n c o d i n g r e g i o n o f m R N A
might
also provide insight into u n d e r s t a n d i n g the translation o f this
m R N A , a n d , in s t u d i e s o f genomic ovalbumin DNA, would identify
the l o c a t i o n o f p o s s i b l e , c o n t i g u o u s " c o n t r o l " sequences,
t h e r e b y a d d i n g to o u r u n d e r s t a n d i n g o f the m e c h a n i s m b y w h i c h
© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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Nucleic Acids Research
e s t r o g e n i n d u c e s t h e s y n t h e s i s o f o v a l b u m i n m R N A in t h e c h i c k
oviduct.
MATERIALS AND METHODS
32
yP-ATP w a s p r e p a r e d by t h e m e t h o d f o r G l y n n and C h a p p e l l
( 4 ) a s d e s c r i b e d by M a x a m a n d G i l b e r t ( 2 ) . D i m e t h y l s u l f a t e a n d
p i p e r i d i n e w e r e p u r c h a s e d from A l d r i c h a n d F i s h e r r e s p e c t i v e l y .
H y d r a z i n e (95%) and imidazole were purchased from Eastman. All
other chemicals were reagent grade.
R e s t r i c t i o n e n d o n u c l e a s e s H i n f I , S s t I, P s t I and T a q I w e r e
purchased from Bethesda Research Labs. T 4 polynucleotide kinase
was purchased from PL Biochemicals o r Boehringer Mannheim.
P O V 2 3 Q D N A w a s p u r i f i e d as d e s c r i b e d by M c R e y n o l d s e_t al .
( 1 ) a n d w a s s u p p l i e d b y D r . A. D u g a i c z y k . p M B 9 , t h e p a r e n t
p l a s m i d of P O V g o g . w a s s u p p l i e d by D r . L . M c R e y n o l d s .
Restriction endonuclease digestions. DNA was digested with
r e s t r i c t i o n e n d o n u c l e a s e s in 6 m M T r i s - H C l (pH 7.5) - 6 m M M g C l 2 6 m M 2 - m e r c a p t o e t h a n o l a t 37°C f o r 3 - 5 h o u r s . D i g e s t i o n m i x t u r e s
w e r e s u p p l e m e n t e d w i t h NaCl as f o l l o w s ; P s t I , 6 m M N a C l ; S s t I,
9 0 m M N a C l ; H i n f I, 2 0 m M N a C l . T a q I d i g e s t i o n s w e r e d o n e a t
5 0 ° C i n 10 m M T r i s - H C l (pH 8.4)-6 m M M g C l 2 - 6 m M 2 - m e r c a p t o e t h a n o l .
F o r p r i m a r y d i g e s t i o n o f total p l a s m i d , a n e n z y m e to D N A r a t i o o f
1-2 u n i t s / p g w a s u s e d . F o r s e c o n d a r y d i g e s t i o n s o f 32 P - l a b e l e d
f r a g m e n t s , ' this ratio w a s 5 units/ug D N A .
5' End l a b e l i n g o f r e s t r i c t i o n f r a g m e n t s .
Double-stranded
DNA r e s t r i c t i o n f r a g m e n t s were
P - l a b e l e d a t t h e 5' e n d o f e a c h
strand using t h e polynucleotide kinase exchange reaction described
32
by B e r k n e r a n d F o l k ( 5 ) . y- P-ATP w a s e v a p o r a t e d to d r y n e s s in a
s i l i c o n i z e d glass tube with a stream of filtered air just prior to
op
use.
The f i n a l
(1500-2000
reaction
Ci/mmole),
dithiothreitol ,
fragments,
The f i n a l
of
the
volume
was
incubated
sulting
P-ATP
(pH 6 . 6 ) ,
Y-
G-50 column
P-ATP
at
37°C
in
o f DNA
to
ten
The r e a c t i o n
a glass
capillary
at
1 0 mM T r i s - H C l
immediately
o f DNA
times
mixture
and t h e
separated
3°C on a 0 . 9
(pH 7 . 5 ) - 1
4 . 5 mM
per pmole
added.
were
by c h r o m a t o g r a p h y
in
pmoles
kinase
volume was a d j u s t e d
kinase
DNA f r a g m e n t s
residual
1 0 uM y -
20-50
T4 p o l y n u c l e o t i d e
reaction
15 m i n u t e s
Sephadex
2254
of
polynucleotide
P-labeled
contained:
3 0 0 uM A D P , 4 5 mM K C 1 , 1 8 mM M g C l 2 ,
25 mM i m i d i z o l e
and 1 u n i t
fragments.
mixture
refrom
x 27 cm
mM E D T A .
The
Nucleic Acids Research
void v o l u m e f r a c t i o n s w e r e c o l l e c t e d and the
P - l a b e l e d DNA p r e c i p i t a t e d a t - 2 0 ° C a f t e r a d d i t i o n of 1/20 v o l u m e of 5 M NaCl and
2 v o l u m e s of e t h a n o l .
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 and r e c o v e r y o f DNA. DNA
r e s t r i c t i o n f r a g m e n t s f r o m p r i m a r y or s e c o n d a r y d i g e s t i o n s w e r e
s e p a r a t e d by e l e c t r o p h o r e s i s in 6% p o l y a c r y l a m i d e s l a b gels ( 1 : 3 0 ,
b i s : a c r y l a m i d e ) c o n t a i n i n g 50 m M T r i s - b o r a t e (pH 8.3) and lmM E D T A .
U n l a b e l e d DNA b a n d s in the gel w e r e s t a i n e d w i t h e t h i d i u m b r o m i d e
(1 u g / m l ) and v i s u a l i z e d u n d e r u l t r a v i o l e t l i g h t .
P - l a b e l e d DNA
f r a g m e n t s w e r e d e t e c t e d by a u t o r a d i o g r a p h y u s i n g D u P o n t C r o n e x 4
32
X-ray film. Some
P - l a b e l e d DNA f r a g m e n t s w e r e s t r a n d s e p a r a t e d
by h e a t d e n a t u r a t i o n (5 m i n u t e s a t 9 0 ° C ) in 5 0 % f o r m a m i d e f o l l o w e d
by r a p i d cool ing to 0 ° C and e l e c t r o p h o r e s i s ( 2 0 0 - 4 0 0 v o l t s ) in an
8% p o l y a ; c r y l a m i d e gel c o n t a i n i n g 7M u r e a - 5 0 mM T r i s - b o r a t e (pH 8.3)
-1 mM E D T A . I n d i v i d u a l DNA b a n d s w e r e c u t o u t o f the gels a n d the
DNA e x t r a c t e d u s i n g the " c r u s h a n d s o a k " t e c h n i q u e d e s c r i b e d by
M a x a m and G i l b e r t ( 2 ) . O p t i m a l DNA r e c o v e r y (>90HS) w a s o b t a i n e d
w i t h a b u f f e r to gel r a t i o ( v / v ) of a t l e a s t 4 : 1 .
DNA s e q u e n c i n g . DNA s e q u e n c i n g w a s p e r f o r m e d e s s e n t i a l l y as
d e s c r i b e d by M a x a m and G i l b e r t ( 2 ) . T h e d i m e t h y l s u l f a t e c l e a v a g e
r e a c t i o n s f o r a d e n i n e and g u a n i n e r e s i d u e s w e r e c a r r i e d out f o r
25 and 15 m i n u t e s r e s p e c t i v e l y . In s o m e c a s e s the a l t e r n a t e Ap r o c e d u r e ( a l k a l i n e c l e a v a g e ) w a s u s e d . H y d r a z i n o l y s i s in t h e
p r e s e n c e ( C - s p e c i f i c ) or a b s e n c e ( p y r i m i d i n e s p e c i f i c ) of 5 M
NaCl w a s c a r r i e d o u t f o r 30 and 20 m i n u t e s r e s p e c t i v e l y . T h e
p r o d u c t s of the i n d i v i d u a l c l e a v a g e r e a c t i o n s w e r e s e p a r a t e d by
e l e c t r o p h o r e s i s in 2 0 % or 1 2 % p o l y a c r y l a m i d e s l a b g e l s c o n t a i n i n g
7 M u r e a , 50 m M T r i s - b o r a t e (pH 8 . 3 ) , and 1 mM E D T A . Two or t h r e e
s u c c e s s i v e l o a d s o f the gel a l l o w e d the u n a m b i g u o u s r e a d i n g o f 6 0 9 0 n u c l e o t i d e s . If a m i n o acid s e q u e n c e d a t a w a s a v a i l a b l e to r e s o l v e a m b i g u i t i e s , as m a n y as 120 n u c l e o t i d e s c o u l d be d e t e r m i n e d .
E a c h r e s t r i c t i o n f r a g m e n t was s e q u e n c e d at l e a s t t w i c e to i n s u r e
a c c u r a c y and e s t a b l i s h o v e r l a p s .
RESULTS
P r e v i o u s w o r k in o u r l a b o r a t o r y ( 3 ) has d e m o n s t r a t e d the r e l a tive location of several restriction endonuclease s i t e s i n double
s t r a n d e d o v a l b u m i n c D N A . The e n z y m e H i n f I w a s of p a r t i c u l a r
i n t e r e s t b e c a u s e it c u t the o v a l b u m i n c D N A ^ j t w i c e in the r e g i o n
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Nucleic Acids Research
expected to contain the AUG initiation codon.
Furthermore, Monahan
et a i . ( 3 ) p r e d i c t e d t h a t this e n z y m e m i g h t a l s o c u t in a r e g i o n
coding for a known ovalbumin peDtide. We therefore began our
s e q u e n c e a n a l y s i s a t t h e H i n f l s i t e s in p O V 2 3 g , t h e c l o n e d f o r m
of c D N A j . C o m p a r i s o n of t h e H i n f I d i g e s t i o n p a t t e r n s o f p a r e n t
plasmid pMB9 and plasmid pOVpTQ. which contains t h e c o m p l e t e o v a l bumin cDNAds, revealed five new, pOV23Q-specific fragments (F2,
F4, F6, F7, and F9) and the disappearance of one of the original
pMB9 fragments (the second largest) (Fig. 1 ) . This missing pMB9
fragment contains the EcoRI site at which ovalbumin cDNA, was
inserted into the plasmid.
F u r t h e r m o r e , the o v a l b u m i n cDNA is
o r i e n t e d in t h e p l a s m i d such that the Hinf I f r a g m e n t c o n t a i n i n g
the 31 end of the cDNA will also contain 65 bp of pMB9 DNA while
the H i n f I f r a g m e n t c o n t a i n i n g t h e cDNA 5' end will c o n t a i n a t
l e a s t 6 2 0 bp o f pMB9 D N A ( 1 , 6 ) .
S i n c e t h e r e is a l s o a n i n t e r n a l
8 0 0 b p H i n f I f r a g m e n t in o v a l b u m i n c D N A d s ( 3 ) , p O V 2 3 Q H i n f I
f r a g m e n t s F 4 a n d F 2 w e r e ordered by t h e i r s i z e a s s h o w n in F i g u r e 3 .
Digestion of
P - l a b e l e d f r a g m e n t s F 6 , F7 a n d F9 w i t h r e s t r i c t i o n
Hinf I
O
<
ro
CM
o
Sst I
CD
to
O
<
ro
01
o
F i g . 1. P r i m a r y r e s t r i c t i o n e n d o n u c l e a s e d i g e s t i o n s . H i n f I
d i g e s t of (a) P O V 2 3 O a n d (t>) p M B 9 , and Sst I d i g e s t of (c) p M B 9
and ( d ) P O V 2 3 Q s e p a r a t e d by 6% 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
and stained with e t h i d i u m b r o m i d e .
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Nucleic Acids Research
endonuclease Pst I revealed that only fragment F7 w a s c u t ( F i g . 2 ) .
S i n c e t h e o n l y P s t I s i t e in o v a l b u m i n c D N A . is a p p r o x i m a t e l y
450 bp from t h e 3' end of the c o d i n g strand ( 3 ) , l o c a t i o n o f
f r a g m e n t F7 on o v a l b u m i n c D N A . w a s d e t e r m i n e d ( F i g . 3 ) . P r e v i o u s
results (3) also indicated that restriction endonuclease Sst I cut
o v a l b u m i n c D N A d s o n l y o n c e a p p r o x i m a t e l y 4 0 0 bp f r o m t h e 31 end of
the cDNA strand; h o w e v e r , Sst I digestion o f pOV«,Q gave two fragm e n t s ( F i g . l ) - o n e 4 0 0 b p l o n g a n d t h e o t h e r t o o l a r g e to enter into t h e 6 % p o l y a c r y l a m i d e g e l . S i n c e p M B 9 c o n t a i n s n o S s t I s i t e s ,
this result indicated the presence of a second Sst I site, very
c l o s e t o t h e 3 ' e n d o f t h e c D N A s t r a n d , in o v a l b u m i n C D N A J . In
a b c d e f g h i
Fig. 2. Secondary digestion of Hinf I restriction fragments from
P°v230F r a g m e n t s F 6 , F 7 , a n d F9 w e r e 5' e n d l a b e l e d w i t h 3 2 p ,
d i g e s t e d w i t h S s t o r P s t a n d e l e c t r o p h o r e s e d o n a 6% p o l y a c r y l a m i d e
s l a b g e l . ( a ) F 6 , ( b ) F 7 , ( c ) F 9 , ( d ) F6 + S s t I, ( e ) F 7 + S s t I ,
( f ) F9 + S s t I, ( g ) F6 + P s t I, ( h ) F7 + P s t I, ( i ) F9 + P s t I.
The black dots i n d i c a t e the p o s i t i o n of the m a r k e r d y e s , x y l e n e
cyanol (=200N) and bromphenol blue ( = 3 5 N ) .
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Nucleic Acids Research
PARTIAL
RESTRICTION
cDNA ov
PMB9
SI
S2
1
51
SI
1
10
5
51
pMB9
Sst I
Sst I
3'
MAP OF pOV23O
F
f
F6
T F7f T
Hint I
Hint I
Hint I
U
15
«
t
F
9
Hinf
F2 31
T
z
Hjnf
Pst I
Fig. 3. Restriction m a p of pOV230 illustrating the relative
p o s i t i o n s o f t h e Hinf I a n d Sst I f r a g m e n t s .
L e n g t h in n u c l e o t i d e s
x T O " 2 is s h o w n b e t w e e n t h e DNA s t r a n d s .
a d d i t i o n , both Hinf
I fragments
be cut by Sst I (Fig.2).
t i o n , it m u s t also c o n t a i n
while
F6 c o n t a i n s
the o v a l b u m i n
the o v a l b u m i n
strand.
(The fragment sizes
of F6 also indicate
cDNAds
a t i t s 3' e n d ) .
(3,7),
the S s t I s i t e o r i g i n a l l y
the second Sst I and therefore
cDNA
Sst I digestion
F6 a n d F 7 , b u t n o t F 9 , w e r e found to
Since the location of F7 was established by Pst I diges-
resulting
from
t h a t t h i s S s t I s i t e is in
s i n c e F6 c a n c o n t a i n o n l y 6 5 b p o f p M B 9 D N A
F i n a l l y , using
f r a g m e n t F 9 w a s located
placement of Hinf
reported ( 3 ) ,
t h e 3' e n d o f
previously mapped
between
I and S s t I sites
sequence analysis also revealed
Hinf I sites
F4 and F2 giving
shown
in F i g u r e
the relative
3.
Initial
t h e p r e s e n c e o f a T a q I s i t e in
f r a g m e n t F6 which w a s subsequently
confirmed
F6 ( d a t a n o t s h o w n ) a n d u s e d f o r s e q u e n c e
by T a q I d i g e s t i o n o f
analysis.
I F i g u r e 4 shows t h e strategy used to d e t e r m i n e t h e entire
sequence of the region of ovalbumin
end o f o v a l b u m i n m e s s e n g e r
well
as fragments generated
labeled
cDNAds
The
restriction
32
P-labeled
endonuclease
by S s t I d i g e s t i o n
by p o l y a c r y l a m i d e
labeled
from F6 w i t h
described
2258
above.
as described
from the Hinf
separated
above.
In t h e
I s i t e in p M B 9
to the T a q I s i t e in o v a l b u m i n c D N A .
5' e n d s w e r e i s o l a t e d
Figure
Sst I) and the result-
at only o n e 5 1 end, w e r e
gel electrophoresis
the A-T linkers
P-labeled
in M a t e r i a l s a n d
DNA f r a g m e n t s w e r e d i g e s t e d w i t h a s e c o n d
c a s e o f t h e F6 s u b f r a g m e n t e x t e n d i n g
two
to t h e 5'
F 6 a n d F7 a s
32
of F6 were
P-
(F6 a n d F 7 w i t h S s t I; SI a n d S 2 w i t h
I; a n d t h e T a q I f r a g m e n t s
ing s u b f r a g m e n t s , e a c h
through
corresponding
Hinf I fragments
a t t h e 5' e n d o f each s t r a n d a s d e s c r i b e d
Methods.
Hinf
RNA.
by strand
, the
separation as
5 shows t h e gel from w h i c h
the nucleotide
Nucleic Acids Research
RESTRICTION SITES USED FOR SEQUENCE ANALYSIS
OF THE OVALBUMIN cDNAds
31
5'CONA
5'
T
mDNA
Taq I
Hinf I
Sst I
T
Hinf I
cDNA
mDNA
Fig. 4 . Expanded r e s t r i c t i o n m a p of pOV23O showing the r e s t r i c t i o n
sites used for sequence a n a l y s i s of the region of POV23O correspondT h e a r r o w s indicate the strands (cDNA
ing to the 5'end of m R N A O v or m D N A ) that w e r e sequenced from their 32p_-| abel e d , 5' ends (*).
cvr c
Fig. 5. DNA s e q u e n c e gel from the f i r s t o v a l b u m i n cDNA<j S H i n f I
site in pOV23OThis sequence reads in the cDNA strand from the
Hinf I site back towards the f i r s t S s t I s i t e .
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Nucleic Acids Research
s e q u e n c e from t h e first H i n f I s i t e in t h e o v a l b u m i n c D N A . back
towards the first Sst I site was determined. As suggested p r e viously ( 3 ) , a known phosphorylated peptide from ovalbumin (8) w a s
e n c o d e d in t h e l o c a t i o n o f t h i s f i r s t H i n f I s i t e . F i g u r e s 6 a n d
'7 s h o w t h e g e l s f r o m w h i c h t h e s e q u e n c e a r o u n d t h e A U G i n i t i a t i o n
codon (as determined from the S s t I and Taq I sites respectively)
w a s d e t e r m i n e d . T h e f i r s t A U G c o d o n lies 52 n u c l e o t i d e s from t h e
A - T l i n k e r s used in c l o n i n g and d i r e c t l y a d j a c e n t to t h e a m i n o
t e r m i n a l peptide o f o v a l b u m i n . There is no i n t e r v e n i n g "signal
C
C*T
6
A
F i g . 6 . D N A s e q u e n c e gel from t h e f i r s t S s t I s i t e in p O V 2 3 O T h i s s e q u e n c e r e a d s in t h e c D N A s t r a n d from t h e f i r s t S s t I s i t e
back towards the AUG initiation codon. The sequence complementary
to t h e i n i t i a t i o n c o d o n ( C A T ) is a l s o s h o w n ( b o x ) .
2260
Nucleic Acids Research
sequence" between
of o v a l b u m i n .
this AUG codon
Finally,
CDNAJ
RNA.
corresponding
by E f s t r a t i a d i s jt^aj.
suggested
nucleotide
after the i n i t i a t i o n
lined amino acids represent
the n u c l e o t i d e
terminal
codon
known
to t h e 5 1
( 9 ) ; that
is n u m b e r o n e .
peptides
glycine
the s e q u e n c e of
The nucleotides are numbered
convention
firming
the a m i n o
Figure 8 illustrates
nucleotides of ovalbumin
ovalbumin messenger
and
end
using
i s , the
The
that were used
302
of
the
first
underin
con-
sequence.
C
C*T
6
A
C
C*T
F i g . 7.
DNA s e q u e n c e gel f r o m t h e T a q I s i t e in p O V 2 3 O This
s e q u e n c e r e a d s in t h e m D N A s t r a n d f r o m the T a q I s i t e t o w a r d s t h e
AUG initiation codon.
The i n i t i a t i o n codon a p p e a r s as A T G ( b o x ) .
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Nucleic Acids Research
Hinf I
(pMB9 D«A)
(Linkers)
3' ICTAAGCAAACCAATTTCCGAAACTCTAAAAGGTCACTGTTTGTACGGTCAGGnCGCTT
5' iGATIflCTTTGGTTAAAGGCTTTGAGATTTTCCAGTGACAAACATGCCAGTCCAAGCGAA
- (A)35- (T)35-
Alul
A l u l TaqI
Hph I
,
,
-40
^ _ , ^ _ "20
1
( c D N A . J TiffCGAfcATAACGGAMTCGTCAGltTCGWGCItTCTGTTGAGTCTC/eGTGa TAC CCG AGG TAG
(mDNA^) AflAiCJETATTGCCTTTAGCAGTC/fefiCfflCiStAGACAACTCAGAGllriAia ATG GGC TCC ATC
fmet qly ser ile
Sst I
3)
40
,
,
CCA CGT CGT TCG TAC CTT AAA ACA AAA CTA CAT AAG TTC ICTC GAS CTT
GGT GCA GCA AGC ATG GAA TTT TGT TTT GAT GTA TTC AAG IGAG CTCj GAA
qly ala ala ser met giu phe cys phe asp vai phe lvs qlu leu lys
Mbo II
60
CAG
GTC
val
Alu I
80
100
GTG GTA CGG TTA CTC TTG 11AG AAfl ATG ACG GGG TAA CGG TAG TAC AST CGPj
CAC CAT GCC AAT GAG AAC flBLUS TAC TGC CCC ATT GCC ATC ATG TCA GCT1
h i s h i s a l a asn q l u asn i l e phe t y r cys p r o i l e a l a i l e met ser a l a
120
140
GAT CGG TAC CAT ATG GAC CCA CGT TTT CTG TCG
CTA GCC ATG GTA TAC CTG GGT GCA AAA GAC AGC
l e u a l a met val t y r leu g l y a l a l y s asp s e r
Eco R I I
180
TTC CAA CAA GCG AAA CTA TTT GAA. |GGT qqr
AAG GTT GTT rar TTT GAT AAA CTT ICCA
lys val val arq phe asp lys leu pro q ly
w
Eco R I I
,
160
TGG TCQ TGT GTT TAT TTA
ACC AGS ACA CAA ATA AAT
t h r arg t h r g i n i l e asn
Hinfl
Alu I
m
AAG CCT CTG TCA TAA CTtT CGA
TTfl r,r,A GAC AGT
phe qly asp ser ile qlu ala
<£)
Hbo I I
240
??!)
GTC ACA CCG TGT AGA CAT TTG CAA GTG AGA AC r GAA
CAG TGT GGC ACA TCT GTA AAC GTT CAC TCT TC CTT
qln cys qly thr ser val asn val his ser ser leu
F i g . 8 . N u c l e o t i d e s e q u e n c e o f o v a l b u m i n cDNA<js c o r r e s p o n d i n g t o
the 5' end o f mRNAov- Also included a r e 5 8 nucleotides from t h e
plasmid pMB9. Known peptides from ovalbumin a r e underlined.
DISCUSSION
From the size o f the ovalbumin c D N A d s insert in pOV~,o and its
ability, when hybridized to ovalbumin messenger RNA, to completely
p r o t e c t t h e m R N A f r o m SI n u c l e a s e d i g e s t i o n , i t i s p o s s i b l e t o
c o n c l u d e that P O V 2 3 O contains a n essentially complete copy o f t h e
ovalbumin mRNA ( 1 ) . T h e 52 nucleotide distance, a s determined b y
nucleotide sequence analysis, o f an AUG codon and adjacent, ovalbumin amino terminal peptide from t h e A - T linkers used in cloning
indicates that t h e 1161 nucleotide coding region o f ovalbumin
m R N A b e g i n s r e l a t i v e l y c l o s e t o t h e 5' e n d o f t h e m o l e c u l e . S i n c e
o v a l b u m i n mRNA contains 1 8 9 0 n u c l e o t i d e s ( 1 0 ) , t h e 3' e n d o f this
m R N A m u s t c o n t a i n m o r e than 6 0 0 n u c l e o t i d e s o f n o n - c o d i n g i n f o r m a t i o n . T h e f u n c t i o n o f this l a r g e , 3 ' n o n - c o d i n g region is p r e s e n t l y
u n k o w n , b u t i t h a s been shown to have some limited sequence homology t o t h e 3' n o n - c o d i n g region o f rabbit globin mRNA ( 1 1 ) .
T h e f i r s t A U G c o d o n in o v a l b u m i n c D N A . i m m e d i a t e l y p r e c e d e s
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Nucleic Acids Research
the codon for the amino terminal glycine of ovalbumin. This result
s t r o n g l y s u g g e s t s that this A U G is the true i n i t i a t i o n c o d o n a n d ,
therefore, that ovalbumin, unlike many other secretory p r o t e i n s ,
d o e s n o t c o n t a i n a " s i g n a l p e p t i d e " . P a l m i t e r e± aJK ( 1 2 ) h a v e
d r a w n s i m i l a r c o n c l u s i o n s by d e t e r m i n i n g t h e a m i n o acid s e q u e n c e of
the 35 a m i n o terminal r e s i d u e s of o v a l b u m i n (See Fig. 8 ) s y n t h e s i z e d
b y t h e J_r^ v i t r o t r a n s l a t i o n o f o v a l b u m i n m R N A . T h e s e i n v e s t i g a t o r s
found that the primary translation product began with m e t h i o n i n e
f o l l o w e d i m m e d i a t e l y by t h e a m i n o t e r m i n a l p e p t i d e of o v a l b u m i n and
t h a t t h e s e q u e n c e o f this p e p t i d e w a s i d e n t i c a l to that of s e c r e t e d
o v a l b u m i n . Most secretory p r o t e i n s , including the other egg w h i t e
proteins c o n a l b u m i n , ovomucoid, and lysozyme (13,14) have been
s h o w n to b e t r a n s l a t e d a s p r e - p r o t e i n s w h i c h c o n t a i n a p p r o x i m a t e l y
20 extra a m i n o a c i d s , most (^80X) of w h i c h a r e h y d r o p h o b i c , at their
a m i n o t e r m i n a l e n d s . T h i s e x t r a p e p t i d e is b e l i e v e d to b e n e c e s s a r y
f o r t h e a t t a c h m e n t o f t h e r i b o s o m e s s y n t h e s i z i n g t h e s e p r o t e i n s to
the endoplasmic reticulum thereby facilitating the vectorial transp o r t o f t h e s e p r o t e i n s t h r o u g h t h e m e m b r a n e r e a u l t i n g in t h e i r
a c c u m u l a t i o n in t h e G o l g i a p p a r a t u s a n d e v e n t u a l s e c r e t i o n ( 1 5 ) .
T h e s i g n a l p e p t i d e is u s u a l l y r e m o v e d d u r i n g m e m b r a n e t r a n s p o r t .
Inspection of the amino terminal region of ovalbumin reveals that
It is p o s s i 12 o f t h e f i r s t 2 0 a m i n o a c i d s ( 6 0 X ) a r e h y d r o p h o b i c .
b l e , t h e r e f o r e , that this region of the protein serves as a "signal
s e q u e n c e " w h i c h r e m a i n s an i n t e g r a l p a r t of t h e m a t u r e p r o t e i n .
A l t e r n a t i v e l y , the acetylation of the amino terminal glycine residue
e a r l y in t h e s y n t h e s i s o f o v a l b u m i n m a y p l a y a r o l e in i t s s e c r e t i o n
(12).
E x a m i n a t i o n of the n u c l e o t i d e s e q u e n c e around the A U G i n i t i a t i o n c o d o n r e v e a l s a p o s s i b l e h a i r p i n l o o p s t r u c t u r e ( F i g u r e 9,
structure A) containing eight base pairs and including the e n t i r e
AUG codon. Using Tinoco's revised rules ( 1 6 ) , the free energy o f
f o r m a t i o n o f t h i s h a i r p i n is e s t i m a t e d a t - 6 . 4 k c a l , s u g g e s t i n g a
stable structure.
Figure 9 also illustrates two possible regions
of c o m p l e m e n t a r i t y between the 5 1 end of o v a l b u m i n mRNA a n d the
highly c o n s e r v e d s e q u e n c e at the 3' end of 18S ribosomal RNA
(17,18).
In " s t r u c t u r e A " , n u c l e o t i d e s f r o m t h e m R N A h a i r p i n
l o o p a r e n o t i n v o l v e d in b i n d i n g t o 1 8 S r R N A ; w h e r e a s , i n " s t r u c t u r e
B " , t h e m R N A h a i r p i n is r e d u c e d to a l e s s s t a b l e , f o u r b a s e p a i r
2263
Nucleic Acids Research
mRNA
A.
A
5' AAAGCUGUAUUGCCUUUGCAGUCAAGCUCGACAGACAACUCAGAGUU
UG
A
CAGCAAGCAUG...
1 8 . , RNA
U
3'A'
G
4;...
5'AAAGCUGUAUUGCCUUUAGCAGUCAAGCUCGACAGACAACUCAGAGUUCACCCGGUGCAGCAAGCAUG-
A G°
I8S rRNA
3'A
F i g . 9. N u c l e o t i d e s e q u e n c e a t 5' e n d o f o v a l b u m i n mRNA s h o w i n g
a possible hairpin loop containing the AUG initiation codon
( s t r u c t u r e A ) a n d p o s s i b l e , complementary base pairing with t h e
3 ' e n d o f 1 8 S r R N A ( s t r u c t u r e s A a n d B ) . In s t r u c t u r e B . , t h e
t w o m{JA-U b a s e p a i r s m a y b e s i m i l a r t o G - U b a s e p a i r s i n t h e i r
hydrogen bonding o r m a y n o t form at all.
s t r u c t u r e (net free e n e r g y = - 1 . 0 kcal) by binding to t h e 1 8 S rRNA.
S i m i l a r possible base pairing h a s been suggested between t h e 3'
end o f 18S rRNA a n d t h e n o n - c o d i n g , 5' ends o f several e u c a r y o t i c
and viral m e s s e n g e r RNAs ( 1 8 ) , h o w e v e r , such i n t e r a c t i o n s a r e
s p e c u l a t i v e a n d t h e i r r o l e , if a n y , i n t h e i n i t i a t i o n o f p r o t e i n
synthesis remains to be determined.
Recent evidence (19,20,21) h a s indicated that t h e coding region
of t h e o v a l b u m i n g e n e is i n t e r r u p t e d a t several points b y
regions of non-coding DNAof varying lengths. Preliminary DNA
s e q u e n c e a n a l y s i s , in o u r laboratory, a t theS s t I a n d Hinf I sites
in g e n o m i c o v a l b u m i n D N A h a s v e r i f i e d t h e p r e s e n c e o f s e v e r a l r e g i o n s
of n o n - c o d i n g , "insert" DNA, indicating a far more complicated
(This data will
s t r u c t u r e than is r e p r e s e n t e d b y o v a l b u m i n c D N A ^ .
be reported elsewhere.)
Finally, using an mRNA template-cDNA primer extension method.
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Nucleic Acids Research
McReynolds e_t aj_. (22) have recently demonstrated the presence of
twelve additional nucleotides at the 5' end of ovalbumin mRNA not
contained in p O V 2 3 o DNA. This region was sequenced and found to
contain no AUG codon, but was capable of forming an eight base
pair, hairpin loop at the 5' end of the mRNA. The sequence data of
these investigators are in essential agreement with the sequence
reported here.
ACKNOWLEDGEMENTS
We gratefully acknowledge Drs. Bert O'Malley and Jeffrey Rosen
for their critical review of this manuscript and Dr. L. McReynolds
and G. Brownlee for providing a copy of their manuscript prior to
publication. This work was supported under NIH grants HD08188-06
and HD07495-05. D.K. is a post-doctoral fellow supported under
NIH training grant HD0444-04.
ABREVIATIONS
=
mRNA
cDNA =
cDNAds rnDNA =
ovalbumin messenger RNA
the complementary DNA copy of a mRNA
double-stranded cDNA
the homologous DNA copy of a mRNA
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