GENOMICS
6, 133-143
(1990)
Direct Sequencing of the Activation Peptide and the Catalytic Domain of the Factor IX Gene in Six Species G. SARKAR, D. D. KOEBERL, AND 5. S. SOMMER’ Department
of Biochemistry
and Molecular Received
Biology, Mayo Clinic/Foundation,
July 19, 1989;
revised
55905
13, 1989
conserved, suggesting that compensatory mutations may have occurred; and (9) when compared to that of mouse, the amino acid identity with guinea pig factor IX is no greater than that found for the non-rodent species, a result compatible with the postulated increased rate of evolution in rodents. 0 1990 Academic
By means of RNA amplification with transcript sequencing (RAWTS) under low stringency conditions, sequence was obtained directly without cloning for the activation peptide and the catalytic domain of factor IX from six species -sheep, pig, rabbit, guinea pig, rat, and mouse. ,The data presented demonstrate that, by the appropriate design of oligonucleotides and by performance of a nested PCR under appropriate conditions, it is possible to obtain sequence on a battery of species with a minimum of oligonucleotide primers. A total of 5.2 kb of cross-species sequence was generated with RAWTS. The results indicate that (1) 69% of the amino acids in the catalytic domain, but only 23% of the amino acids in the activation pepticle, are identical in humans and the six species; (2) the catalytic domain evolves at a slower rate, but the extent and pattern of conservation of amino acids in the activation peptide suggest that the pepticle functions as more than a cleavage spacer that separates the heavy and light chains in the catalytically inactive zymogen; (3) 37% of the amino acids in the activation peptide and 34% of the amino acids in the catalytic domain are factor XX-speciific; i.e., they are either identical or changed in a highly conservative fashion in factor IX, but not in other related coagulation proteaeee; (4) these conserved factor IX-specific amino acids fall into three clusters, which are candidates for involvement in the protein interactions specific to factor IX; (5) there is a human-specific deletion after lysine 142 and a rodent-specific insertion after alanine 161; (6) in guinea pig, the insertion is associated with a sevenamino-acid repeat that corresponds to a perfect repeat of a 21-bp sequence; (7) humans have lost a potential N-glycosylation site that is conserved in the other species; (8) in each species, a few nonconservative changes occur in amino acids that are otherwise completely Sequence data from this article have been deposited EMBL/GenBank Data Libraries under Accession Nos. M26234, M26235, M26236, M26237, and M23247. ’ To whom reprint requests should be addressed.
September
Rochester, Minnesota
Press,
Inc.
INTRODUCTION
Human factor IX is an activatable serine coagulation protease that is encoded by a 34-kb gene on the X chromosome (Yoshitake et al., 1985). Factor IX circulates in the plasma as a single-chain, vitamin Kdependent zymogen of 415 amino acids that contains 17% carbohydrate by weight. During clotting, factor IX may be activated by factor XIa in the presence of calcium and by factor VIIa in the presence of calcium and tissue factor (reviewed in Furie and Furie, 1988). Proteolysis by either factor XIa or factor VIIa releases a 35-amino-acid activation peptide. The primary role of activated factor IX in clotting is to activate factor X. The physiological complex includes factor IXa, calcium, phospholipid, and thrombin-activated factor VIII. Factor IXa also binds to anti-thrombin III and it can activate factor VII in the presence of calcium and phospholipid (DiScipio et al., 1978; Masys et al., 1982). DNA sequence for the coding region of factor IX is available for humans (Kurachi and Davie, 1982; Jaye et al., 1983; Anson et al., 1984; Jagadeeswaran et al., 1984; McGraw et al., 1985) and the amino acid sequence is available for the circulating zymogen from cow (Katayama et al., 1979). If additional sequenceswere available, it would be possible to better define the conserved amino acids. A subset of these amino acids will be generic in the sensethat they are also conserved in factor VII, factor X, and protein C-a group of related coagulation proteases that have the same domains and identical exonic structures (Furie and Furie, 1988). The remainder of the conserved amino acids will be unique
with the M26233,
133
0888-7543/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
134
SARKAR.
K OEBERL,
AND 1
A TGC
SOMMER 2
3
4
5
\ ATGC(ATGCIATGCIATGC
FIG. 1. (A) Amplification of the activation peptide and the catalytic domain from multiple species. PCR was performed on liver cDNA with primers to human sequence complementary to the beginning of exon F and the end of the coding region in exon H (OliA and B). If there was no major insertion or deletion, an approximately 900-bp segment migrating just above the third largest size standard was expected. Lane S, size standards produced by Hoe111 digestion of @X174; lane N, control amplification with no template DNA; lane 1, mouse cDNA, lane 2, rat cDNA; lane 3, guinea pig cDNA, lane 4, rabbit cDNA, and lane 5, sheep cDNA. (B) Sequence of a segment from mouse (l), rat (2), guinea pig (3), rabbit (4), and sheep (5). Sequence was obtained by RAWTS using the PCR primer (OliB) as the sequencing primer.
to factor IX. These will be candidates for specific interactions of factor IX with factors VII, VIII, X, XI, and anti-thrombin III. ZooRAWTS is a technique that in principle allows the sequence of homologs in multiple speciesto be obtained rapidly without the need for cloning (Sarkar and Sommer, 1989). Here we demonstrate more extensively the feasibility of ZooRAWTS by obtaining over 5 kb of DNA sequencefrom the activation and catalytic domains of the factor IX from six species. The data define amino acids that are specific for factor IX. The catalytic domain of factor IX is found to be highly conserved and the activation domain also has a substantial fraction of conserved amino acids, strongly suggesting that it functions as more than just an inactivating spacer between the heavy and light chains. MATERIALS
Liver RAWTS
AND
METHODS
mRNAs were purchased from Clontech. was performed as indicated below.
1. First-strand cDNA synthesis: Twenty microliters of 50 pg/ml heat-denatured total RNA or mRNA, 50 m&f Tris-HCl (pH 8.3), 8 mA4 magnesium chloride, 30 n&f KCl, 1 n&f DTT, 2 r&f each dATP, dCTP, dGTP, dTTP, 50 @g/ml oligo(dT) 12-18,lOOO U/ml RNasin, and 1000 U/ml AMV reverse transcriptase were incubated at 42“C for 1 h, followed by 65°C for 10 min. Subsequently, 30 ~1 of Hz0 was added, generating a final volume of 50 ~1.
2. PCR: The above sample (1~1) was added to 40 ~1 of 50 n-& KCl, 10 mM Tris-HCl (pH 8.3), 2.0-8.0 n&f MgC12 (empirically determined for each set of primers), 0.01% (w/v) gelatin, 200 & each dNTP, 1 plkl each primer (Perkin-Elmer-Cetus protocol). After 10 min at 94”C, 1 U of Taq polymerase was added and 30-40 cycles of PCR were performed (denaturation: 1 min at 94’C; annealing: 2 min at 5O’C; elongation: 3 min at 72°C) with the Perkin-Elmer-Cetus automated thermal cycler. One primer included a T7 or SP6 promoter
DIRECT
SEQUENCING
TABLE Mismatches
Compatible Obtaining
Species and olieonucleotide I. OliD Human Sheep Pig Rabbit Guinea Rat Mouse II. OliF Human Sheep Pig Rabbit Guinea Rat Mouse III.
OliJ Human Sheep Pig Rabbit Guinea Rat Mouse
pig
pig
pig
OF
IX
ACTIVATION
1 and Incompatible Sequence
Seauence
FACTOR
with
T ............ ....... A ................... .................... ..G..........T ...... .G.....T.....T ...... c ..... 5' &&tiiibTACTGA3' ..A ............. 5' TGCTGCATTCTGTGGA 3' ..A .......... ..C ..AG..G ......... ..AA ............ ..AG ............ ..AG ............ ..G..........GC.C.. .. . A.....CTC.. ..G........C..CG.C. . . . . . . . . . . . . . . CG.. ..G....C...C...GA.. .. .. .. .. . ... . .. ... . 5' ACATAGCTGTTTAGTATTA3'
+ + +
Nomenclature
as previously described (Stoflet et al., 1988). After the last cycle of PCR, a final lo-min elongation was performed.
FIG. 2. differences.
Sequencing Tbe arrows
strategy indicate
2oov
3oov
4oov
135
DOMAINS
4. Sequencing protocol [modification of Geliebter (1987)]: The transcription reaction (2 ~1) was added to 10 ~1 of annealing buffer containing the end-labeled reverse transcriptase primer. Annealing and sequencing were performed essentially as described (Stoflet et al., 1988), except that lessreverse transcriptase (2.5 units/ reaction tube) was used. Note that [T-~~P]ATP is the correct donor for end-labeling.
a The oligonucleotide sequence is shown and differences between the oligonucleotide sequence and the species sequence are indicated OliJ is in the upstream direction so the sequence is complementary to the sequence in Fig. 3. b PCR was performed using the indicated oligonucleotide and either OliA or OliC. Less than 10 pg of the 9Wbp T7-SP6 segment was the input DNA. If an amplification product of appropriate size was seen, it was eluted from an agarose gel, transcribed, and sequenced with the PCR oligonucleotide.
1OOV
6, 133-143
(1990)
Direct Sequencing of the Activation Peptide and the Catalytic Domain of the Factor IX Gene in Six Species G. SARKAR, D. D. KOEBERL, AND 5. S. SOMMER’ Department
of Biochemistry
and Molecular Received
Biology, Mayo Clinic/Foundation,
July 19, 1989;
revised
55905
13, 1989
conserved, suggesting that compensatory mutations may have occurred; and (9) when compared to that of mouse, the amino acid identity with guinea pig factor IX is no greater than that found for the non-rodent species, a result compatible with the postulated increased rate of evolution in rodents. 0 1990 Academic
By means of RNA amplification with transcript sequencing (RAWTS) under low stringency conditions, sequence was obtained directly without cloning for the activation peptide and the catalytic domain of factor IX from six species -sheep, pig, rabbit, guinea pig, rat, and mouse. ,The data presented demonstrate that, by the appropriate design of oligonucleotides and by performance of a nested PCR under appropriate conditions, it is possible to obtain sequence on a battery of species with a minimum of oligonucleotide primers. A total of 5.2 kb of cross-species sequence was generated with RAWTS. The results indicate that (1) 69% of the amino acids in the catalytic domain, but only 23% of the amino acids in the activation pepticle, are identical in humans and the six species; (2) the catalytic domain evolves at a slower rate, but the extent and pattern of conservation of amino acids in the activation peptide suggest that the pepticle functions as more than a cleavage spacer that separates the heavy and light chains in the catalytically inactive zymogen; (3) 37% of the amino acids in the activation peptide and 34% of the amino acids in the catalytic domain are factor XX-speciific; i.e., they are either identical or changed in a highly conservative fashion in factor IX, but not in other related coagulation proteaeee; (4) these conserved factor IX-specific amino acids fall into three clusters, which are candidates for involvement in the protein interactions specific to factor IX; (5) there is a human-specific deletion after lysine 142 and a rodent-specific insertion after alanine 161; (6) in guinea pig, the insertion is associated with a sevenamino-acid repeat that corresponds to a perfect repeat of a 21-bp sequence; (7) humans have lost a potential N-glycosylation site that is conserved in the other species; (8) in each species, a few nonconservative changes occur in amino acids that are otherwise completely Sequence data from this article have been deposited EMBL/GenBank Data Libraries under Accession Nos. M26234, M26235, M26236, M26237, and M23247. ’ To whom reprint requests should be addressed.
September
Rochester, Minnesota
Press,
Inc.
INTRODUCTION
Human factor IX is an activatable serine coagulation protease that is encoded by a 34-kb gene on the X chromosome (Yoshitake et al., 1985). Factor IX circulates in the plasma as a single-chain, vitamin Kdependent zymogen of 415 amino acids that contains 17% carbohydrate by weight. During clotting, factor IX may be activated by factor XIa in the presence of calcium and by factor VIIa in the presence of calcium and tissue factor (reviewed in Furie and Furie, 1988). Proteolysis by either factor XIa or factor VIIa releases a 35-amino-acid activation peptide. The primary role of activated factor IX in clotting is to activate factor X. The physiological complex includes factor IXa, calcium, phospholipid, and thrombin-activated factor VIII. Factor IXa also binds to anti-thrombin III and it can activate factor VII in the presence of calcium and phospholipid (DiScipio et al., 1978; Masys et al., 1982). DNA sequence for the coding region of factor IX is available for humans (Kurachi and Davie, 1982; Jaye et al., 1983; Anson et al., 1984; Jagadeeswaran et al., 1984; McGraw et al., 1985) and the amino acid sequence is available for the circulating zymogen from cow (Katayama et al., 1979). If additional sequenceswere available, it would be possible to better define the conserved amino acids. A subset of these amino acids will be generic in the sensethat they are also conserved in factor VII, factor X, and protein C-a group of related coagulation proteases that have the same domains and identical exonic structures (Furie and Furie, 1988). The remainder of the conserved amino acids will be unique
with the M26233,
133
0888-7543/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
134
SARKAR.
K OEBERL,
AND 1
A TGC
SOMMER 2
3
4
5
\ ATGC(ATGCIATGCIATGC
FIG. 1. (A) Amplification of the activation peptide and the catalytic domain from multiple species. PCR was performed on liver cDNA with primers to human sequence complementary to the beginning of exon F and the end of the coding region in exon H (OliA and B). If there was no major insertion or deletion, an approximately 900-bp segment migrating just above the third largest size standard was expected. Lane S, size standards produced by Hoe111 digestion of @X174; lane N, control amplification with no template DNA; lane 1, mouse cDNA, lane 2, rat cDNA; lane 3, guinea pig cDNA, lane 4, rabbit cDNA, and lane 5, sheep cDNA. (B) Sequence of a segment from mouse (l), rat (2), guinea pig (3), rabbit (4), and sheep (5). Sequence was obtained by RAWTS using the PCR primer (OliB) as the sequencing primer.
to factor IX. These will be candidates for specific interactions of factor IX with factors VII, VIII, X, XI, and anti-thrombin III. ZooRAWTS is a technique that in principle allows the sequence of homologs in multiple speciesto be obtained rapidly without the need for cloning (Sarkar and Sommer, 1989). Here we demonstrate more extensively the feasibility of ZooRAWTS by obtaining over 5 kb of DNA sequencefrom the activation and catalytic domains of the factor IX from six species. The data define amino acids that are specific for factor IX. The catalytic domain of factor IX is found to be highly conserved and the activation domain also has a substantial fraction of conserved amino acids, strongly suggesting that it functions as more than just an inactivating spacer between the heavy and light chains. MATERIALS
Liver RAWTS
AND
METHODS
mRNAs were purchased from Clontech. was performed as indicated below.
1. First-strand cDNA synthesis: Twenty microliters of 50 pg/ml heat-denatured total RNA or mRNA, 50 m&f Tris-HCl (pH 8.3), 8 mA4 magnesium chloride, 30 n&f KCl, 1 n&f DTT, 2 r&f each dATP, dCTP, dGTP, dTTP, 50 @g/ml oligo(dT) 12-18,lOOO U/ml RNasin, and 1000 U/ml AMV reverse transcriptase were incubated at 42“C for 1 h, followed by 65°C for 10 min. Subsequently, 30 ~1 of Hz0 was added, generating a final volume of 50 ~1.
2. PCR: The above sample (1~1) was added to 40 ~1 of 50 n-& KCl, 10 mM Tris-HCl (pH 8.3), 2.0-8.0 n&f MgC12 (empirically determined for each set of primers), 0.01% (w/v) gelatin, 200 & each dNTP, 1 plkl each primer (Perkin-Elmer-Cetus protocol). After 10 min at 94”C, 1 U of Taq polymerase was added and 30-40 cycles of PCR were performed (denaturation: 1 min at 94’C; annealing: 2 min at 5O’C; elongation: 3 min at 72°C) with the Perkin-Elmer-Cetus automated thermal cycler. One primer included a T7 or SP6 promoter
DIRECT
SEQUENCING
TABLE Mismatches
Compatible Obtaining
Species and olieonucleotide I. OliD Human Sheep Pig Rabbit Guinea Rat Mouse II. OliF Human Sheep Pig Rabbit Guinea Rat Mouse III.
OliJ Human Sheep Pig Rabbit Guinea Rat Mouse
pig
pig
pig
OF
IX
ACTIVATION
1 and Incompatible Sequence
Seauence
FACTOR
with
T ............ ....... A ................... .................... ..G..........T ...... .G.....T.....T ...... c ..... 5' &&tiiibTACTGA3' ..A ............. 5' TGCTGCATTCTGTGGA 3' ..A .......... ..C ..AG..G ......... ..AA ............ ..AG ............ ..AG ............ ..G..........GC.C.. .. . A.....CTC.. ..G........C..CG.C. . . . . . . . . . . . . . . CG.. ..G....C...C...GA.. .. .. .. .. . ... . .. ... . 5' ACATAGCTGTTTAGTATTA3'
+ + +
Nomenclature
as previously described (Stoflet et al., 1988). After the last cycle of PCR, a final lo-min elongation was performed.
FIG. 2. differences.
Sequencing Tbe arrows
strategy indicate
2oov
3oov
4oov
135
DOMAINS
4. Sequencing protocol [modification of Geliebter (1987)]: The transcription reaction (2 ~1) was added to 10 ~1 of annealing buffer containing the end-labeled reverse transcriptase primer. Annealing and sequencing were performed essentially as described (Stoflet et al., 1988), except that lessreverse transcriptase (2.5 units/ reaction tube) was used. Note that [T-~~P]ATP is the correct donor for end-labeling.
a The oligonucleotide sequence is shown and differences between the oligonucleotide sequence and the species sequence are indicated OliJ is in the upstream direction so the sequence is complementary to the sequence in Fig. 3. b PCR was performed using the indicated oligonucleotide and either OliA or OliC. Less than 10 pg of the 9Wbp T7-SP6 segment was the input DNA. If an amplification product of appropriate size was seen, it was eluted from an agarose gel, transcribed, and sequenced with the PCR oligonucleotide.
1OOV
