Degenerate PCR method for identification of an antiapoptotic gene in BHV-1.
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 gamma34.5, 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.
Marfè
G
G
Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata-Via Montpellier 1, 00133 Rome, Italy.
De Martino
L
L
Filomeni
G
G
Di Stefano
C
C
Giganti
M G
MG
Pagnini
U
U
Napolitano
F
F
Iovane
G
G
Ciriolo
M R
MR
Salimei
P Sinibaldi
PS
D020816:Amino Acid Motifs; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D016384:Consensus Sequence; D017931:DNA Primers / Q000737:chemistry; D019992:Databases as Topic; D050884:HSP72 Heat-Shock Proteins / Q000235:genetics; D007242:Herpesvirus 1, Bovine / Q000235:genetics*; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D012984:Software; D014764:Viral Proteins / Q000235:genetics* / Q000502:physiology
D017931:DNA Primers; D050884:HSP72 Heat-Shock Proteins; C402639:UL14 protein, Human herpesvirus 1; D014764:Viral Proteins