.=) 1990 Oxford University Press
5564 Nucleic Acids Research, Vol. 18, No. 18
Gene sequence of porcine tumor necrosis factor alpha Roman T.Drews, Bradford W.Coffee, Annie K.Prestwood' and Royal A.McGraw* Departments of Physiology and Pharmacology and 1Parasitology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA EMBL accession no. X54001
Submitted August 3, 1990 We report genomic DNA sequence for porcine tumor necrosis factor alpha. The gene segment was obtained by PCR (1) from porcine genomic DNA using the primers: 5' -GCGGATCCATGAGCACTGAAAGCATGATC -3' (sense) and 5 '-GCCGAATTCAGGGCAATGATCCCAAAGT-3' (antisense). Amplification was performed in a homemade thermal cycling apparatus (2). Primers were based on published human sequence (3) from the extremes of known coding regions. Restriction sites were included at the 5' ends as indicated (underlined). A PCR product of 1.8 kb was excised and purified from a .7% agarose gel, cleaved with EcoRI and BamHI, cloned into pUC19, and further subcloned into M13 vectors for conventional dideoxy sequencing in both strands. The resulting sequence (BamHI-EcoRI fragment, 1806 bp) is shown in Figure 1. Coding regions of this sequence differ from the PCR-derived partial cDNA sequence of Pauli and co-workers (4) at three positions: C not T at 1657, A not G at 1768, and T not C at 1771. Regions corresponding to 136-156 and -
1766-1802 of this sequence were primer-derived in the sequence of Pauli and co-workers. Curiously, the 3' nucleotide (G) of their sense primer for cDNA amplification differed from their reported cDNA sequence (an A corresponding to our position 156). None of these differences alter the predicted translation, however.
ACKNOWLEDGEMENT This work was supported by the University of Georgia Program in Biological Resources and Biotechnology.
REFERENCES 1. Saiki,R.K., Gelfand,D.H., Stoffel,S., Scharf,S.J., Higuchi,R., Horn,G.T., Mullis,K.B. and Ehrlich,H.A. (1988) Science 239, 487-491. 2. McGraw,R.A., Steffe,E.K. and Hutchinson,C.A. (1988) DNA and Protein Engineering Techniques 1, 65-67. 3. Shirai,T., Yamaguchi,H., Ito,H., Todd,C.W. and Wallace,R.B. (1985) Nature 313, 803-806. 4. Pauli,U., Beutler,B. and Peterhans,E. (1989) Gene 81, 185-191.
1701
T E S M S M I R D V E L A E E A L A K K A G G P Q G S R R C L C GAC GCACT; CATGATCCGA GACGTGGAGC TGGCGGAGGA GGCGCTCGCC AAGAAGGCCG GGGGCCCCCA GGGCTCCAGG AGGTGCCTGT 100 F S F L S L L L V A G A T T L F C L L H F E V I G P Q K E E GCCTCAGCCT CTTCTCCTTC CTCCTGGTCG CAGGAGCCAC CACGCTCTTC TGCCTACTGC ACTTCGAGGT TATCGGCCCC CAGAAGGAAG AG GTGAGCGC 200 CTGGCCAGCC GTGGCTCATT CTCCCACCCG GAGAGAAATG GGAAGAAAGA GGGCCGAGGA CGAGCTGGGG GAAAGAAGTG TGCTGATGGG GAGTGTGGGG 300 AGGAAATCAT GGAGAAAGAT GGGGAGGCAG AAGGAGACGT GGAGACACAT GGGGGGAGAG AGAGAAGGAT GGAGAGAAAT CCGGTGCCCG CCCTTGGAAA 400 TGCTCTCTAA ATATTTGTTG CACGAATGAG TGAGTGAGCA GGGACACTGA TAGAAAGAGA GATGAGTAGA CAGACAAGGG GTGTGGTAGA AAGATAGGGA 500 AAAAACAAGT GATCCGGATA AAGATAGTGA GACAGGAAGA GGTAGAGGAG ATAGGAAAGA GAGATAAGGA GAGAGAAGAA GAAGGAAGCG TGGGTGTCTG 600 GCACGTGGAA GGCACTCAAT GAAAGAGTTG TTGAATGGAT GGGTGGATGA GAAAATGGAT GAGTGGAGAG AAAAAACTAG ACATCAGGGC AGAGAGTACA 700 AGCTAGAGAA GCAGGTGGTG TTTTCCCTTC AGAGGGGACT TATTCAAATC TAATTAATCC TCCTTCTTCT CCCCAACAG F P A G P L S T TTCCAGCTGG CCCCTTGAGC 800 I N P L A Q G L R ATCAACCCTC TGGCCCAAGG ACTCA GTAAG TATCTCTAAA ACCTGTCTCT CAGTTCTGAG CTTGGACAGG GGTGGGGTTA GTGCTGGGGT GGAAGGAAGA 900 AGGGAAATTT AGGGTCTGGG TTTGGCGGGG GGGAATGCAG GGCTCAAAGT AGTGAGATAT TTTCTGGGAA GTCTGAGGGT CTCATCTTTT TCTTTCCTCT 1000 TTCCTCCTCA G Q T S S S S D K P V A H V V A GATCATCGT CTCAAACCTC AGATAAGCCC GTCGCCCACG TTGTAG GTAA GAGTTCTGAG GATGTGTGTC TGGGGGATGA AGAAATAGGC 1100 AGGACAGAGA GGGATAGGAT TTGGGGCTGA AGCCAGGCTG AGGGTAGCCA GAGCTTGGAG ATAGTATGAG GAGGACTCGC TGAGCTCCAG GGGAGGATGG 1200 GGGATACTCA GAACTTGAGG AGGATACTCG GAACCTCATG GACAGATGGG ATGTGGGAAG ACAGACCGAG GGGACAGGAA CCGGATGTGG GGGGCGGGCA 1300 GAACTCGAGG GCCAGGATGT GGAGAGTGGA ACTGGACAGG GTCACACTGA CTQCCCCCTC CCTCTTTGTC TCCTCCCTCC AG N V K A E G CCAATGTC AAAGCCGAGG 1400 G Y A N A L L Q L Q W Q S A N G V K L K D N Q L V V P T D G L Y L GACAGCTCCA GTGGCAGAGT GGGTATGCCA ATGCCCTCCT GGCCAACGGC GTGAAGCTGA AAGACAACCA GCTGGTGGTG CCGACAGATG GGCTGTACCT 1500 I Y S Q V L F R G Q G C P S T N V F L T H T I S R I A V S Y Q T K CATCTACTCC CAGGTCCTCT TCAGGGGCCA AGGCTGCCCT TCACCAACG TTTTCCTCAC TCACACCATC AGCCGCATCG CCGTCTCCTA CCAGACCAAG 1600 V N L L S A I K S P C Q R E T P E G A E A K P W Y E P I Y L G G V F GTCAACCTCC TCTCTGCCAT CAAGAGCCCT TGCCAGAGGG AGACCCCCGA GGGGGCCGAG GCCAAGCCCT GGTACGAACC CATCTACCTG GGAGGGGTCT 1700 K D D Q L E R L S A E I N L P D Y L D F A E S G Q V Y F G I I A L TCCAGCTGGA GAAGGATGAT CGACTCAGTG CCGAGATCAA CCTGCCCGAC TATCTGGACT TTGCTGAATC TGGGCAGGTC TCATTCCCT 1800
1801
fA&iCTr
1
101 201 301 401 501 601 701
801
901 1001
1101 1201 1301
1401 1501
1601
*
TA-TTTIC&
1806
Figure 1. Nucleotide sequence of gene for porcine tumor necrosis factor alpha. Predicted translation is shown above the nucleotide sequence. Primer-derived sequences
are underlined. *
To whom correspondence should be addressed
5564 Nucleic Acids Research, Vol. 18, No. 18
Gene sequence of porcine tumor necrosis factor alpha Roman T.Drews, Bradford W.Coffee, Annie K.Prestwood' and Royal A.McGraw* Departments of Physiology and Pharmacology and 1Parasitology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA EMBL accession no. X54001
Submitted August 3, 1990 We report genomic DNA sequence for porcine tumor necrosis factor alpha. The gene segment was obtained by PCR (1) from porcine genomic DNA using the primers: 5' -GCGGATCCATGAGCACTGAAAGCATGATC -3' (sense) and 5 '-GCCGAATTCAGGGCAATGATCCCAAAGT-3' (antisense). Amplification was performed in a homemade thermal cycling apparatus (2). Primers were based on published human sequence (3) from the extremes of known coding regions. Restriction sites were included at the 5' ends as indicated (underlined). A PCR product of 1.8 kb was excised and purified from a .7% agarose gel, cleaved with EcoRI and BamHI, cloned into pUC19, and further subcloned into M13 vectors for conventional dideoxy sequencing in both strands. The resulting sequence (BamHI-EcoRI fragment, 1806 bp) is shown in Figure 1. Coding regions of this sequence differ from the PCR-derived partial cDNA sequence of Pauli and co-workers (4) at three positions: C not T at 1657, A not G at 1768, and T not C at 1771. Regions corresponding to 136-156 and -
1766-1802 of this sequence were primer-derived in the sequence of Pauli and co-workers. Curiously, the 3' nucleotide (G) of their sense primer for cDNA amplification differed from their reported cDNA sequence (an A corresponding to our position 156). None of these differences alter the predicted translation, however.
ACKNOWLEDGEMENT This work was supported by the University of Georgia Program in Biological Resources and Biotechnology.
REFERENCES 1. Saiki,R.K., Gelfand,D.H., Stoffel,S., Scharf,S.J., Higuchi,R., Horn,G.T., Mullis,K.B. and Ehrlich,H.A. (1988) Science 239, 487-491. 2. McGraw,R.A., Steffe,E.K. and Hutchinson,C.A. (1988) DNA and Protein Engineering Techniques 1, 65-67. 3. Shirai,T., Yamaguchi,H., Ito,H., Todd,C.W. and Wallace,R.B. (1985) Nature 313, 803-806. 4. Pauli,U., Beutler,B. and Peterhans,E. (1989) Gene 81, 185-191.
1701
T E S M S M I R D V E L A E E A L A K K A G G P Q G S R R C L C GAC GCACT; CATGATCCGA GACGTGGAGC TGGCGGAGGA GGCGCTCGCC AAGAAGGCCG GGGGCCCCCA GGGCTCCAGG AGGTGCCTGT 100 F S F L S L L L V A G A T T L F C L L H F E V I G P Q K E E GCCTCAGCCT CTTCTCCTTC CTCCTGGTCG CAGGAGCCAC CACGCTCTTC TGCCTACTGC ACTTCGAGGT TATCGGCCCC CAGAAGGAAG AG GTGAGCGC 200 CTGGCCAGCC GTGGCTCATT CTCCCACCCG GAGAGAAATG GGAAGAAAGA GGGCCGAGGA CGAGCTGGGG GAAAGAAGTG TGCTGATGGG GAGTGTGGGG 300 AGGAAATCAT GGAGAAAGAT GGGGAGGCAG AAGGAGACGT GGAGACACAT GGGGGGAGAG AGAGAAGGAT GGAGAGAAAT CCGGTGCCCG CCCTTGGAAA 400 TGCTCTCTAA ATATTTGTTG CACGAATGAG TGAGTGAGCA GGGACACTGA TAGAAAGAGA GATGAGTAGA CAGACAAGGG GTGTGGTAGA AAGATAGGGA 500 AAAAACAAGT GATCCGGATA AAGATAGTGA GACAGGAAGA GGTAGAGGAG ATAGGAAAGA GAGATAAGGA GAGAGAAGAA GAAGGAAGCG TGGGTGTCTG 600 GCACGTGGAA GGCACTCAAT GAAAGAGTTG TTGAATGGAT GGGTGGATGA GAAAATGGAT GAGTGGAGAG AAAAAACTAG ACATCAGGGC AGAGAGTACA 700 AGCTAGAGAA GCAGGTGGTG TTTTCCCTTC AGAGGGGACT TATTCAAATC TAATTAATCC TCCTTCTTCT CCCCAACAG F P A G P L S T TTCCAGCTGG CCCCTTGAGC 800 I N P L A Q G L R ATCAACCCTC TGGCCCAAGG ACTCA GTAAG TATCTCTAAA ACCTGTCTCT CAGTTCTGAG CTTGGACAGG GGTGGGGTTA GTGCTGGGGT GGAAGGAAGA 900 AGGGAAATTT AGGGTCTGGG TTTGGCGGGG GGGAATGCAG GGCTCAAAGT AGTGAGATAT TTTCTGGGAA GTCTGAGGGT CTCATCTTTT TCTTTCCTCT 1000 TTCCTCCTCA G Q T S S S S D K P V A H V V A GATCATCGT CTCAAACCTC AGATAAGCCC GTCGCCCACG TTGTAG GTAA GAGTTCTGAG GATGTGTGTC TGGGGGATGA AGAAATAGGC 1100 AGGACAGAGA GGGATAGGAT TTGGGGCTGA AGCCAGGCTG AGGGTAGCCA GAGCTTGGAG ATAGTATGAG GAGGACTCGC TGAGCTCCAG GGGAGGATGG 1200 GGGATACTCA GAACTTGAGG AGGATACTCG GAACCTCATG GACAGATGGG ATGTGGGAAG ACAGACCGAG GGGACAGGAA CCGGATGTGG GGGGCGGGCA 1300 GAACTCGAGG GCCAGGATGT GGAGAGTGGA ACTGGACAGG GTCACACTGA CTQCCCCCTC CCTCTTTGTC TCCTCCCTCC AG N V K A E G CCAATGTC AAAGCCGAGG 1400 G Y A N A L L Q L Q W Q S A N G V K L K D N Q L V V P T D G L Y L GACAGCTCCA GTGGCAGAGT GGGTATGCCA ATGCCCTCCT GGCCAACGGC GTGAAGCTGA AAGACAACCA GCTGGTGGTG CCGACAGATG GGCTGTACCT 1500 I Y S Q V L F R G Q G C P S T N V F L T H T I S R I A V S Y Q T K CATCTACTCC CAGGTCCTCT TCAGGGGCCA AGGCTGCCCT TCACCAACG TTTTCCTCAC TCACACCATC AGCCGCATCG CCGTCTCCTA CCAGACCAAG 1600 V N L L S A I K S P C Q R E T P E G A E A K P W Y E P I Y L G G V F GTCAACCTCC TCTCTGCCAT CAAGAGCCCT TGCCAGAGGG AGACCCCCGA GGGGGCCGAG GCCAAGCCCT GGTACGAACC CATCTACCTG GGAGGGGTCT 1700 K D D Q L E R L S A E I N L P D Y L D F A E S G Q V Y F G I I A L TCCAGCTGGA GAAGGATGAT CGACTCAGTG CCGAGATCAA CCTGCCCGAC TATCTGGACT TTGCTGAATC TGGGCAGGTC TCATTCCCT 1800
1801
fA&iCTr
1
101 201 301 401 501 601 701
801
901 1001
1101 1201 1301
1401 1501
1601
*
TA-TTTIC&
1806
Figure 1. Nucleotide sequence of gene for porcine tumor necrosis factor alpha. Predicted translation is shown above the nucleotide sequence. Primer-derived sequences
are underlined. *
To whom correspondence should be addressed
