THROMBOSIS RESEARCH 59; 389-393,199O 0049-3848/90 $3.00 + .OO Printed in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.
BRIEF
EVIDENCE
COMMUNICATION
FOR A GLYCINE RESIDUE AT POSITION
Department
(Received
316 IN HUMAN PROTEIN C INHIBITOR.
JOOST C.M. MEIJERS and DOMINIC W. CHUNG of Biochemistry SJ-70, University of Washington, Seattle, WA 98195, U.S.A.
7.3.1990;
accepted
in revised form 1.5.1990
by Editor G. Vehar)
INTRODUCTION Protein C inhibitor' (PCI) is a single-chain glycoprotein with a molecular weight of 57,000 (1). It is one of the two proteinase inhibitors involved in the regulation of activated protein C (2-4). The amino acid sequence of human PC1 was deduced from the cDNA sequence described by Suzuki et al. (5). In our efforts to characterize the cDNA of PCI, a difference was found with the published sequence at position 316; GGC (Gly) was found instead of CGC (Arg). This sequence predicts the presence of a Msp I cleavage site. Amplification of the genomic region with the polymerase chain reaction followed by restriction digest with Msp I and comparison of the same region with other members of the serpin family indicated that this discrepancy is most likely caused by a rare polymorphism, or a cloning or sequencing artefact.
MATERIALS
AND METHODS
One pair of oligonucleotides (PJ-1 and PJ-2) was used to amplify the cDNA of PC1 from a liver library, kindly provided by Dr. S.L.C. Woo (Baylor College, 6), and a Hep G2 cell library (7). The polymerase chain reaction (PCR) was performed as recommended by Perkin-Elmer/Cetus. The annealing temperature was 45 "C. The cycle was repeated 30 times followed by digestion with Barn HI. The digested product was cloned into M13mp18 for sequencing. The cDNAs were sequenced by the dideoxy chain termination method (8) using Ml3 universal primers and PCI-specific oligonucleotide primers. Oligonucleotides were synthesized on an Applied Biosystems oligonucleotide synthesizer (Foster City, CA). A different set of oligonucleotides (PJ-4 and PJ-5) was used to confirm the amplified cDNA sequence. Genomic DNA of 20 Seattle Caucasians (kindly provided by Dr. Jonathan Tait, University of Washington) was amplified by PCR. The annealing temperature was 55 "C, and the cycle was repeated 30 times. The
Key words: protein
C inhibitor,
cDNA, amino acid sequence
389
390
SEQUENCE OF PROTEIN C INHIBITOR
Vol. 69, No. 2
3' C
T
-/
Ile
A C G *G C C
Gly 316
ser
T G
\
T
Leu 314
C 5’
Figure 1. Sequence of the PC1 cDNA region encoding aa314-317. base discrepancy is marked by an asterisk.
The single
_-
154 142,075
--135 --lo6
1--
std.
+
cDNA
-
+
Genomic DNA
Figure 2. Agarose gel electrophoresis of amplified regions of cDNA and genomic DNA before (-) and after digestion with Msp I (+).
Vol. 59, No. 2
SEQUENCE OF PROTEIN C INHIBITOR
391
product was then subjected to Msp I restriction digest and electrophoresed on a 1.5% agarose gel (BRL) containing ethidium bromide.
TABLE I: Oligonucleotide Primers used in the PCR Reaction. PJ-1: PJ-2: PJ-4: PJ-5:
TTTGGATCCCTCATAGAACAAAGAACATCCACC TTTGGATCCGTAGATTTCAGGAGAAGCCCCACC CGAGCTTTACCTTCCCAAATTCTCC ACCTGGATATTTGAGTGGTTGCTG
Primers are in 5'-3' orientation. BarnHI site is underlined.
RESULTS AN'DDISCUSSION A pair of oligonucleotide primers (PJ-1 and PJ-2, Table I) was used to amplify the cDNA for human PC1 from two different Xgtll libraries, a liver library and a Hep G2 cell library. The primers were constructed to anneal prior to the initiator methionine, and after the stop-codon (5), thereby amplifying the complete coding region of 1.3 kb. The primers contained Barn HI restriction site extensions to facilitate cloning and sequencing. Upon sequencing of the PCR-products from both libraries, a single difference was observed with the previously described sequence of PC1 (5). Figure 1 shows that the codon GGC for glycine was observed instead of CGC for arginine at position 316. This predicts the presence of a restriction site for Msp I (CCGG). To investigate if the discrepancy was due to a polymorphism, a 135 bp fragment of PC1 was amplified from the genomic DNA of 20 Seattle Caucasians using two oligonucleotide primers (PJ-4 and PJ-5, Table I). Both the cDNA, and the genomic DNA show 135 bp bands before Msp I digest (Figure 2). With the genomic DNA sample a non-specific band (55 bp) is also observed. After digestion with the restriction enzyme Msp I, the 135 bp band is cut into two pieces of 106 and 29. bp in length. This digestion pattern was observed in the amplified products of all the 20 genomic DNA's (data not shown). These results indicate that glycine was presen; in position 316'of all the individuals examined, and suggest that the original described sequence is a rare polymorphism (
BRIEF
EVIDENCE
COMMUNICATION
FOR A GLYCINE RESIDUE AT POSITION
Department
(Received
316 IN HUMAN PROTEIN C INHIBITOR.
JOOST C.M. MEIJERS and DOMINIC W. CHUNG of Biochemistry SJ-70, University of Washington, Seattle, WA 98195, U.S.A.
7.3.1990;
accepted
in revised form 1.5.1990
by Editor G. Vehar)
INTRODUCTION Protein C inhibitor' (PCI) is a single-chain glycoprotein with a molecular weight of 57,000 (1). It is one of the two proteinase inhibitors involved in the regulation of activated protein C (2-4). The amino acid sequence of human PC1 was deduced from the cDNA sequence described by Suzuki et al. (5). In our efforts to characterize the cDNA of PCI, a difference was found with the published sequence at position 316; GGC (Gly) was found instead of CGC (Arg). This sequence predicts the presence of a Msp I cleavage site. Amplification of the genomic region with the polymerase chain reaction followed by restriction digest with Msp I and comparison of the same region with other members of the serpin family indicated that this discrepancy is most likely caused by a rare polymorphism, or a cloning or sequencing artefact.
MATERIALS
AND METHODS
One pair of oligonucleotides (PJ-1 and PJ-2) was used to amplify the cDNA of PC1 from a liver library, kindly provided by Dr. S.L.C. Woo (Baylor College, 6), and a Hep G2 cell library (7). The polymerase chain reaction (PCR) was performed as recommended by Perkin-Elmer/Cetus. The annealing temperature was 45 "C. The cycle was repeated 30 times followed by digestion with Barn HI. The digested product was cloned into M13mp18 for sequencing. The cDNAs were sequenced by the dideoxy chain termination method (8) using Ml3 universal primers and PCI-specific oligonucleotide primers. Oligonucleotides were synthesized on an Applied Biosystems oligonucleotide synthesizer (Foster City, CA). A different set of oligonucleotides (PJ-4 and PJ-5) was used to confirm the amplified cDNA sequence. Genomic DNA of 20 Seattle Caucasians (kindly provided by Dr. Jonathan Tait, University of Washington) was amplified by PCR. The annealing temperature was 55 "C, and the cycle was repeated 30 times. The
Key words: protein
C inhibitor,
cDNA, amino acid sequence
389
390
SEQUENCE OF PROTEIN C INHIBITOR
Vol. 69, No. 2
3' C
T
-/
Ile
A C G *G C C
Gly 316
ser
T G
\
T
Leu 314
C 5’
Figure 1. Sequence of the PC1 cDNA region encoding aa314-317. base discrepancy is marked by an asterisk.
The single
_-
154 142,075
--135 --lo6
1--
std.
+
cDNA
-
+
Genomic DNA
Figure 2. Agarose gel electrophoresis of amplified regions of cDNA and genomic DNA before (-) and after digestion with Msp I (+).
Vol. 59, No. 2
SEQUENCE OF PROTEIN C INHIBITOR
391
product was then subjected to Msp I restriction digest and electrophoresed on a 1.5% agarose gel (BRL) containing ethidium bromide.
TABLE I: Oligonucleotide Primers used in the PCR Reaction. PJ-1: PJ-2: PJ-4: PJ-5:
TTTGGATCCCTCATAGAACAAAGAACATCCACC TTTGGATCCGTAGATTTCAGGAGAAGCCCCACC CGAGCTTTACCTTCCCAAATTCTCC ACCTGGATATTTGAGTGGTTGCTG
Primers are in 5'-3' orientation. BarnHI site is underlined.
RESULTS AN'DDISCUSSION A pair of oligonucleotide primers (PJ-1 and PJ-2, Table I) was used to amplify the cDNA for human PC1 from two different Xgtll libraries, a liver library and a Hep G2 cell library. The primers were constructed to anneal prior to the initiator methionine, and after the stop-codon (5), thereby amplifying the complete coding region of 1.3 kb. The primers contained Barn HI restriction site extensions to facilitate cloning and sequencing. Upon sequencing of the PCR-products from both libraries, a single difference was observed with the previously described sequence of PC1 (5). Figure 1 shows that the codon GGC for glycine was observed instead of CGC for arginine at position 316. This predicts the presence of a restriction site for Msp I (CCGG). To investigate if the discrepancy was due to a polymorphism, a 135 bp fragment of PC1 was amplified from the genomic DNA of 20 Seattle Caucasians using two oligonucleotide primers (PJ-4 and PJ-5, Table I). Both the cDNA, and the genomic DNA show 135 bp bands before Msp I digest (Figure 2). With the genomic DNA sample a non-specific band (55 bp) is also observed. After digestion with the restriction enzyme Msp I, the 135 bp band is cut into two pieces of 106 and 29. bp in length. This digestion pattern was observed in the amplified products of all the 20 genomic DNA's (data not shown). These results indicate that glycine was presen; in position 316'of all the individuals examined, and suggest that the original described sequence is a rare polymorphism (
