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Degenerate PCR method for identification of an antiapoptotic gene in BHV‐1
Author(s) -
Marfè G.,
De Martino L.,
Filomeni G.,
Di Stefano C.,
Giganti M.G.,
Pagnini U.,
Napolitano F.,
Iovane G.,
Ciriolo M.R.,
Salimei P. Sinibaldi
Publication year - 2005
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.20636
Subject(s) - gene , microbiology and biotechnology , biology , genomic dna , dna , heat shock protein , oligonucleotide , genetics
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.