Journal: Journal of Virology
Article Title: The Region between Amino Acids 245 and 265 of the Bovine Leukemia Virus (BLV) Tax Protein Restricts Transactivation Not Only via the BLV Enhancer but Also via Other Retrovirus Enhancers
Figure Lengend Snippet: Transactivation of the BLV LTR by various tax clones from BLV-infected cattle. (A and B) BLV tax genes were amplified by PCR with EX Taq (A) or KOD-Plus (B) DNA polymerase from the genomic DNAs of BLV-infected animals and from a molecular clone pBLV-IF and then subcloned into pME18Neo. 293T cells were transfected with the reporter plasmid pGV-BLTR, the reference plasmid pRL-SV40, and individual Tax-expressing plasmids. At 60 h after transfection, cells were recovered and the activities of firefly and Renilla luciferases were measured in lysates. For each sample, the firefly luciferase activity (pGV-BLTR) was normalized by reference to Renilla luciferase activity (pRL-SV40). The extent of transactivation (fold) was calculated by dividing the transactivation activity of the mutant Tax protein by that of wild-type Tax protein. (C) Summary of the results shown in panels A and B. The tax mutant clones were divided into three groups as follows, with wild-type activity being taken as 1: high, > 3-fold transactivation; medium, 3- to 0.5-fold transactivation; low,
Article Snippet: Approximately 10 to 100 ng of genomic or plasmid DNA was amplified in a 30-μl reaction mixture that contained 1× EX Taq buffer (Takara Shuzo, Ohtsu, Japan) or 1× KOD-Plus buffer containing 1 mM MgSO4 , (Toyobo, Kyoto, Japan), 0.25 mM each deoxynucleoside triphosphate, 0.33 μM each oligonucleotide primer, and 1 U of EX Taq (Takara Shuzo) or KOD-Plus™ (Toyobo) DNA polymerase.
Techniques: Clone Assay, Infection, Amplification, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Mutagenesis