Degenerate PCR method for identification of an antiapoptotic gene in BHV-1

✍ Scribed by G. Marfè; L. De Martino; G. Filomeni; C. Di Stefano; M.G. Giganti; U. Pagnini; F. Napolitano; G. Iovane; M.R. Ciriolo; P. Sinibaldi Salimei

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 209 KB
• Volume: 97
• Category: Article
• ISSN: 0730-2312
• DOI: 10.1002/jcb.20636

✦ Synopsis

Abstract

To investigate on the hypothetical presence of an antiapoptotic gene, we utilized the CODEHOP (COnsensus‐DEgenerate Hybrid Oligonucleotide Primers) strategy amplifying unknown sequences from a background of genomic (bovine herpesvirus type‐1) BHV‐1 DNA. An alignment of carboxyl‐terminal domains belonging to three proteins encoded by γ34.5, MyD116 and GADD34 genes, was carried out to design degenerate PCR primers in highly conserved regions. This allowed the amplification of a 110 bp fragment. This fragment was subjected to automatic sequencing and DNA sequence analysis revealed that its position resided between the nt 14363 and the nt 14438 in bovine herpesvirus type‐1 (BHV‐1) Cooper strain sharing an identity of 86% (UL14). Transient transfections showed that UL14 protein is efficient in protecting MDBK and K562 cells from sorbitol induced apoptosis. The protein's anti‐apoptotic function may derive from its heat shock protein‐like properties. J. Cell. Biochem. 97: 813–823, 2006. © 2005 Wiley‐Liss, Inc.