Porcine tumor necrosis factor alpha: cloning with the polymerase chain reaction and determination of the nucleotide sequence.
We have cloned the gene for the porcine tumor necrosis factor alpha (TNF-alpha) utilizing the polymerase chain reaction (PCR). Total RNA from stimulated monocytes was used to generate TNF-alpha-specific single-stranded cDNA, with reverse transcriptase and a conserved consensus primer, starting at the TNF-alpha translation stop codon. After adding a second conserved consensus primer to the propeptide region, we amplified the cDNA between the two primers. The isolated fragment was cloned and sequenced. Comparison of the nucleotide sequence indicated an 85% sequence similarity to the human TNF-alpha gene. Eighteen amino acids of the deduced mature peptide sequence of porcine TNF-alpha were different from those in the sequence of man. The technique used in this work allows rapid cloning of specific genes from total RNA, and, additionally, screens for full-length transcripts during the amplification procedure.
Pauli
U
U
Institute for Veterinary Virology, University of Berne, Switzerland.
Beutler
B
B
Peterhans
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000096:biosynthesis*; D004587:Electrophoresis, Agar Gel; D005784:Gene Amplification; D008969:Molecular Sequence Data; D009000:Monocytes / Q000032:analysis; D010446:Peptide Fragments / Q000235:genetics; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics*
D004277:DNA, Single-Stranded; D010446:Peptide Fragments; D014409:Tumor Necrosis Factor-alpha; D012313:RNA