Porcine tumor necrosis factor alpha: cloning with the polymerase chain reaction and determination of the nucleotide sequence

Urs Pauli, Bruce Beutler, Ernst Peterhans

Research output: Contribution to journalArticle

36 Scopus citations

Abstract

We have cloned the gene for the porcine tumor necrosis factor α (TNF-α.) utilizing the polymerase chain reaction (PCR). Total RNA from stimulated monocytes was used to generate TNF-α-specific single-stranded cDNA, with reverse transcriptase and a conserved consensus primer, starting at the TNF-α. 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-α gene. Eighteen amino acids of the deduced mature peptide sequence of porcine TNF-α 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.

Original languageEnglish (US)
Pages (from-to)185-191
Number of pages7
JournalGene
Volume81
Issue number1
DOIs
StatePublished - Sep 1 1989

Keywords

  • Recombinant DNA
  • amino acid sequence homology
  • amplification
  • cachectin
  • complementary DNA
  • evolution

ASJC Scopus subject areas

  • Genetics

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