Nucleic Acids Research, Vol. 18, No. 14 4251
©D 1990 Oxford University Press
cDNA
sequence
of rat prothrombin
Melitta Dihanich and Denis Monard Friedrich Miescher Institute, Basel, Switzerland Submitted June 13, 1990
EMBL accession
mRNA isolated from rat liver was reverse transcribed. From the resulting cDNAs (1 ng) a 500 bp fragment of rat prothrombin was amplified by the polymerase chain reaction (PCR) using oligonucleotide primers made according to sequences conserved in serine proteases (1) (sense primer: 5' GGTGCGCA/CTT/GGGC/AA/GAGACAC 3', antisense primer: 5 FA/CG/AGGT/GCCA/cCCAC/GT/AGTCAC 3'). This PCRfragment served as a hybridization probe to isolate cDNA clones from a rat liver library in Xzapll (Stratagene). The largest cDNA encodes an open reading frame of 1851 bp and has 80.9% identity with the human sequence (2). A comparison of rat, human and bovine protein sequences shows that the three glycosylation sites mapped in bovine prothrombin (3) are conserved in rat; moreover rat prothrombin has an additional potential glycosylation site at asparagine 552. The sequence is registered with EMBL (Accession no. X52835) (4).
discussions.
REFERENCES 1. Furie,B., Bing,D.H., Feldmann,R.J., Robinson,D.J., Buirnier,J.P. and Furie,B.C. (1982) J. Biol. Chem. 257, 3875-3882. 2. Friezner-Degen,S.J. and Davie,E.W. (1987) Biochemistry 26, 6165-6177. 3. Davie,E.W., Fujikawa,K., Kurachi,K. and Kisiel,W. (1979) Adv. Enzym. Relat. Areas Mol. Biol. 48, 277-318. 4. Cameron,G.N. (1988) Nucd. Acids Res. 16, 1865-1867.
-80 GGCTGTGGTGACCGGAACAAGCCCGGGGTCTACACTGACGTGGCCAATTACCTGGATTAGATCCCAGGAGCTGACACACT
-1
90
M
L
H
V
R
G
L
G
L
P
G
C
L
A
L
A
A
L
A
S
L
V
H
S Q
H
V
F
L
A
30
P
Q
Q
A
L
S
L
L
Q
R
V
R
R
A
N
S
G
F
L
E
E
L
R
K
N
L
E
R
E
180 60
G
TGCGTGGAAGAGCAGTGCAGCTATGAGGAGGCTTTTGAGGCCCTGGAGTCTCCCCAAGACACGGATGTGTTCTGGGCCAAGTACACAGTG V
E
E
Q
C
S
Y
E
E
A
F
E
A
L
E
S
P
Q
D
T
D
V
F
W
A
K
Y
T
V
TGTGATTCAGTGAGGAAACCTCGAGAGACCTTCATGGACTGTCTAGAAGGTCGCTGTGCTATGGATCTGGGTCTGAACTACCATGGGAAT C
D
S
V
R
K
P
R
E
T
F
M
D
C
L
E G
R
C
A
M
D
L
G
L
N
Y
H
G
N
GTGAGTGTCACCCATACCGGCATCGAGTGTCAGCTGTGGCGAAGCCGCTACCCACACAGGCCTGACATCAACTCCACCACCCATCCTGGG V S V T H T G I E C Q L W R S R Y P H R P D I N S T T H P G GCTGACCTGAAGGAGAACTTCTGCCGAAATCCAGACAGCAGTACCTCAGGGCCCTGGTGCTACACCACAGACCCTACTGTGCGCAGAGAG
270 90 360 120 450 150 540 180
A D L K E N F C R N P D S S T S G P W C Y T T D P T V R R E GAATGCAGTATCCCTGTCTGTGGTCAGGAGGGCCGTACCACTGTGAAAATGACTCCTCGCTCAAGAGGTTCCAAGGAGAATCTGTCGCCT 630 E C S I P V C G 0 E G R T T V K M T P R S R G S K E N L S P 210 CCATTGGGAGAGTGCCTCCTCGAGCGTGGCCGGCTGTACCAGGGGAACTTAGCTGTGACAACACTTGGGTCCCCCTGCCTGGCCTGGGAC 720 P L G E C L L E R G R L Y Q G N L A V T T L G S P C L A W D 240 810 AGTTTGCCAACCAAGACCCTGAGCAAGTACCAGAACTTCGACCCTGAGGTGAAACTCGTGCAAAACTTCTGCCGCAATCCAGACCGGGAT S L P T K T L S K Y 0 N F D P E V K L V Q N F C R N P D R D 270 900 GAGGAGGGCGCGTGGTGCTTTGTTGCGCAGCAGCCTGGTTTTGAATACTGCAGCCTCAACTACTGTGATGAAGCGGTGGGAGAGGAGAAC E E G A W C F V A 0 0 P G F E Y C S L N Y C D E A V G E E N 300 CATGACGGGGACGAGTCCATCGCAGGACGCACCACCGACGCGGAGTTCCACACCTTCTTCGATGAGAGGACTTTCGGCCTTGGGGAGGCA 990 H D G D E S I A G R T T D A E F H T F F D E R T F G L G E A 330 GACTGTGGCCTGCGGCCTTTGTTCGAGAAGAAGTCCTTGACAGACAAAACGGAAAAGGAGCTTCTTGATTCTTACATAGATGGGCGCATC 1080 D C G L R P L F E K K S L T D K T E K E L L D S Y I D G R I 360 GTGGAGGGCTGGGACGCTGAGAAGGGTATCGCCCCATGGCAGGTGATGCTTTTTCGGAAGAGTCCCCAAGAGCTGCTGTGTGGAGCCAGC 1170 V E G W D A E K G I A P W Q V M L F R K S P Q E L L C G A S 390 CTTATCAGTGATCGATGGGTCCTCACTGCTGCCCACTGCATTCTGTACCCACCCTGGGACAAGAACTTCACTGAGAATGACCTCCTGGTG 1260 420 L I S D R W V L T A A H C I L Y P P W D K N F T E N D L L V CGCATTGGCAAGCACTCCAGAACCAGATATGAGCGGAATGTTGAAAAGATCTCCATGCTGGAAAAGATCTACATCCACCCCAGATATAAC 1350 450 R I G K H S R T R Y E R N V E K I S M L E K I Y I H P R Y N TGGCGGGAAAACCTAGACCGTGACATTGCTCTGCTCAAGCTAAAGAAGCCAGTGCCCTTCAGTGACTACATTCACCCCGTGTGCTTGCCA 1440 W
R
E
N
L
D
R
D
I
A
L
L
K
L
K
K
P
V
P
F
S
D
Y
I
H
P
V
C
L
P
480
GACAAGCAGACAGTAACCAGTTTGCTCCAGGCTGGTTATAAAGGGCGGGTGACAGGCTGGGGCAACCTTCGGGAGACGTGGACAACCAAC 1530 510 D K Q T V T S L L 0 A G Y K G R V T G W G N L R E T W T T N ATCAACGAGATACAGCCCAGCGTCCTGCAGGTGGTGAACCTGCCCATTGTAGAGCGACCAGTGTGCAAGGCCTCTACCCGGATACGCATT 1620 540 I N E I Q P S V L Q V V N L P I V E R P V C K A S T R I R I
ACTGACAACATGTTCTGTGCTGGCTTCAAGGTGAATGACACCAAGCGAGGAGATGCTTGTGAAGGTGACAGTGGGGGACCTTTTGTCATG 1710 T
D
N
M
F
C
A
G
F
K
V
N
D
T
K
R
G
D
A
C
E
G
D
S
G
G
P
F
V
M
570
AAGAGCCCCTACAACCACCGCTGGTACCAAATGGGTATTGTCTCCTGGGGTGAAGGCTGTGACCGGAATGGGAAATATGGCTTCTACACG 1800 K
S
P
Y
N
H
R
W
Y
Q
M
G
I
V
S
W
G
E
G
C
D
R
N
G
K
Y
G
F
Y
T
600
CACGTGTTCCGTCTGAAAAGGTGGATGCAGAAAGTCATTGATCAGCATAGATAGGTTTGTGGGTGGGTGAGAGACCCACAGTATGGGATC 1890 H
V
F
R
L
K
R
W
M
Q
K
V
I
D
Q
X52835
ACKNOWLEDGEMENTS We would like to thank Sara Kozma for the rat liver cDNA library, Werner Zurcher and Franz Fischer for synthesis of the oligonucleotides, Christoph Nager, Martin Spiess, and Jurg Spring for help with the computer and Hanspeter Saluz for helpful
ATGTTGCACGTCCGCGGCCTGGGGCTACCTGGCTGCCTGGCTCTGGCTGCCCTGGCTAGCCTGGTACACAGTCAGCATGTGTTCCTGGCC
C
no.
H
R
*
617
CTCATTGCAAAATCACAGAAACCAGTTCAAAATATTTTTGTGGTTTGTTTCTGTTCTCAATAAAAGTGATTGTCG 1965
©D 1990 Oxford University Press
cDNA
sequence
of rat prothrombin
Melitta Dihanich and Denis Monard Friedrich Miescher Institute, Basel, Switzerland Submitted June 13, 1990
EMBL accession
mRNA isolated from rat liver was reverse transcribed. From the resulting cDNAs (1 ng) a 500 bp fragment of rat prothrombin was amplified by the polymerase chain reaction (PCR) using oligonucleotide primers made according to sequences conserved in serine proteases (1) (sense primer: 5' GGTGCGCA/CTT/GGGC/AA/GAGACAC 3', antisense primer: 5 FA/CG/AGGT/GCCA/cCCAC/GT/AGTCAC 3'). This PCRfragment served as a hybridization probe to isolate cDNA clones from a rat liver library in Xzapll (Stratagene). The largest cDNA encodes an open reading frame of 1851 bp and has 80.9% identity with the human sequence (2). A comparison of rat, human and bovine protein sequences shows that the three glycosylation sites mapped in bovine prothrombin (3) are conserved in rat; moreover rat prothrombin has an additional potential glycosylation site at asparagine 552. The sequence is registered with EMBL (Accession no. X52835) (4).
discussions.
REFERENCES 1. Furie,B., Bing,D.H., Feldmann,R.J., Robinson,D.J., Buirnier,J.P. and Furie,B.C. (1982) J. Biol. Chem. 257, 3875-3882. 2. Friezner-Degen,S.J. and Davie,E.W. (1987) Biochemistry 26, 6165-6177. 3. Davie,E.W., Fujikawa,K., Kurachi,K. and Kisiel,W. (1979) Adv. Enzym. Relat. Areas Mol. Biol. 48, 277-318. 4. Cameron,G.N. (1988) Nucd. Acids Res. 16, 1865-1867.
-80 GGCTGTGGTGACCGGAACAAGCCCGGGGTCTACACTGACGTGGCCAATTACCTGGATTAGATCCCAGGAGCTGACACACT
-1
90
M
L
H
V
R
G
L
G
L
P
G
C
L
A
L
A
A
L
A
S
L
V
H
S Q
H
V
F
L
A
30
P
Q
Q
A
L
S
L
L
Q
R
V
R
R
A
N
S
G
F
L
E
E
L
R
K
N
L
E
R
E
180 60
G
TGCGTGGAAGAGCAGTGCAGCTATGAGGAGGCTTTTGAGGCCCTGGAGTCTCCCCAAGACACGGATGTGTTCTGGGCCAAGTACACAGTG V
E
E
Q
C
S
Y
E
E
A
F
E
A
L
E
S
P
Q
D
T
D
V
F
W
A
K
Y
T
V
TGTGATTCAGTGAGGAAACCTCGAGAGACCTTCATGGACTGTCTAGAAGGTCGCTGTGCTATGGATCTGGGTCTGAACTACCATGGGAAT C
D
S
V
R
K
P
R
E
T
F
M
D
C
L
E G
R
C
A
M
D
L
G
L
N
Y
H
G
N
GTGAGTGTCACCCATACCGGCATCGAGTGTCAGCTGTGGCGAAGCCGCTACCCACACAGGCCTGACATCAACTCCACCACCCATCCTGGG V S V T H T G I E C Q L W R S R Y P H R P D I N S T T H P G GCTGACCTGAAGGAGAACTTCTGCCGAAATCCAGACAGCAGTACCTCAGGGCCCTGGTGCTACACCACAGACCCTACTGTGCGCAGAGAG
270 90 360 120 450 150 540 180
A D L K E N F C R N P D S S T S G P W C Y T T D P T V R R E GAATGCAGTATCCCTGTCTGTGGTCAGGAGGGCCGTACCACTGTGAAAATGACTCCTCGCTCAAGAGGTTCCAAGGAGAATCTGTCGCCT 630 E C S I P V C G 0 E G R T T V K M T P R S R G S K E N L S P 210 CCATTGGGAGAGTGCCTCCTCGAGCGTGGCCGGCTGTACCAGGGGAACTTAGCTGTGACAACACTTGGGTCCCCCTGCCTGGCCTGGGAC 720 P L G E C L L E R G R L Y Q G N L A V T T L G S P C L A W D 240 810 AGTTTGCCAACCAAGACCCTGAGCAAGTACCAGAACTTCGACCCTGAGGTGAAACTCGTGCAAAACTTCTGCCGCAATCCAGACCGGGAT S L P T K T L S K Y 0 N F D P E V K L V Q N F C R N P D R D 270 900 GAGGAGGGCGCGTGGTGCTTTGTTGCGCAGCAGCCTGGTTTTGAATACTGCAGCCTCAACTACTGTGATGAAGCGGTGGGAGAGGAGAAC E E G A W C F V A 0 0 P G F E Y C S L N Y C D E A V G E E N 300 CATGACGGGGACGAGTCCATCGCAGGACGCACCACCGACGCGGAGTTCCACACCTTCTTCGATGAGAGGACTTTCGGCCTTGGGGAGGCA 990 H D G D E S I A G R T T D A E F H T F F D E R T F G L G E A 330 GACTGTGGCCTGCGGCCTTTGTTCGAGAAGAAGTCCTTGACAGACAAAACGGAAAAGGAGCTTCTTGATTCTTACATAGATGGGCGCATC 1080 D C G L R P L F E K K S L T D K T E K E L L D S Y I D G R I 360 GTGGAGGGCTGGGACGCTGAGAAGGGTATCGCCCCATGGCAGGTGATGCTTTTTCGGAAGAGTCCCCAAGAGCTGCTGTGTGGAGCCAGC 1170 V E G W D A E K G I A P W Q V M L F R K S P Q E L L C G A S 390 CTTATCAGTGATCGATGGGTCCTCACTGCTGCCCACTGCATTCTGTACCCACCCTGGGACAAGAACTTCACTGAGAATGACCTCCTGGTG 1260 420 L I S D R W V L T A A H C I L Y P P W D K N F T E N D L L V CGCATTGGCAAGCACTCCAGAACCAGATATGAGCGGAATGTTGAAAAGATCTCCATGCTGGAAAAGATCTACATCCACCCCAGATATAAC 1350 450 R I G K H S R T R Y E R N V E K I S M L E K I Y I H P R Y N TGGCGGGAAAACCTAGACCGTGACATTGCTCTGCTCAAGCTAAAGAAGCCAGTGCCCTTCAGTGACTACATTCACCCCGTGTGCTTGCCA 1440 W
R
E
N
L
D
R
D
I
A
L
L
K
L
K
K
P
V
P
F
S
D
Y
I
H
P
V
C
L
P
480
GACAAGCAGACAGTAACCAGTTTGCTCCAGGCTGGTTATAAAGGGCGGGTGACAGGCTGGGGCAACCTTCGGGAGACGTGGACAACCAAC 1530 510 D K Q T V T S L L 0 A G Y K G R V T G W G N L R E T W T T N ATCAACGAGATACAGCCCAGCGTCCTGCAGGTGGTGAACCTGCCCATTGTAGAGCGACCAGTGTGCAAGGCCTCTACCCGGATACGCATT 1620 540 I N E I Q P S V L Q V V N L P I V E R P V C K A S T R I R I
ACTGACAACATGTTCTGTGCTGGCTTCAAGGTGAATGACACCAAGCGAGGAGATGCTTGTGAAGGTGACAGTGGGGGACCTTTTGTCATG 1710 T
D
N
M
F
C
A
G
F
K
V
N
D
T
K
R
G
D
A
C
E
G
D
S
G
G
P
F
V
M
570
AAGAGCCCCTACAACCACCGCTGGTACCAAATGGGTATTGTCTCCTGGGGTGAAGGCTGTGACCGGAATGGGAAATATGGCTTCTACACG 1800 K
S
P
Y
N
H
R
W
Y
Q
M
G
I
V
S
W
G
E
G
C
D
R
N
G
K
Y
G
F
Y
T
600
CACGTGTTCCGTCTGAAAAGGTGGATGCAGAAAGTCATTGATCAGCATAGATAGGTTTGTGGGTGGGTGAGAGACCCACAGTATGGGATC 1890 H
V
F
R
L
K
R
W
M
Q
K
V
I
D
Q
X52835
ACKNOWLEDGEMENTS We would like to thank Sara Kozma for the rat liver cDNA library, Werner Zurcher and Franz Fischer for synthesis of the oligonucleotides, Christoph Nager, Martin Spiess, and Jurg Spring for help with the computer and Hanspeter Saluz for helpful
ATGTTGCACGTCCGCGGCCTGGGGCTACCTGGCTGCCTGGCTCTGGCTGCCCTGGCTAGCCTGGTACACAGTCAGCATGTGTTCCTGGCC
C
no.
H
R
*
617
CTCATTGCAAAATCACAGAAACCAGTTCAAAATATTTTTGTGGTTTGTTTCTGTTCTCAATAAAAGTGATTGTCG 1965
