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 gamma 34.5, 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 ULI 4 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.
Degenerate PCR method for identification of an antiapoptotic gene in BHV-1 / G., Marfe; DE MARTINO, Luisa; G., Filomeni; C., DI STEFANO; M. G., Giganti; Pagnini, Ugo; F., Napolitano; Iovane, Giuseppe; M. R., Ciriolo; P., SINIBALDI SALIMEI. - In: JOURNAL OF CELLULAR BIOCHEMISTRY. - ISSN 0730-2312. - STAMPA. - 97:4(2006), pp. 813-823. [10.1002/jcb.20636]
Degenerate PCR method for identification of an antiapoptotic gene in BHV-1.
DE MARTINO, LUISA;PAGNINI, UGO;IOVANE, GIUSEPPE;
2006
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 gamma 34.5, 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 ULI 4 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
