Five different point mutations were generated by site-directed mutagenesis PCR at codon 143, 175, 248, 249 or 273.
A pCMV6-AC expression vector containing the MICA gene (Origene, Rockville, ML, USA) was altered by site-directed mutagenesis at two positions (codons for amino acids 24 and 360) to obtain the MICA*00701 allele.
This construct was used as a template for PCR-based mutagenesis of the catalytic domain at codon 273 (D91N: GAT to AAT) and codon 303 (E101Q: GAG to CAG).
By in vitro mutagenesis, it was shown that a mutation at codon D588N of UL54 conferred 9-fold reduced susceptibility to PFA, while a mutation at codon V781I induced 4-fold reduced susceptibility to PFA and GCV.
This sequence encodes a phenylalanine at codon 47 and site directed mutagenesis using the QuickChange protocol [49] was used to change codon 47 to one that encodes leucine.
Indeed, we found that co-injection of Myc-def but not of Myc-def stop (a def mutant that harbors a premature stop codon at codon 55 created by site-directed mutagenesis) mRNA drastically reduced the level of p53 protein but not that of p53 mRNA at 6 h post-injection (hpi).
21 pCY4B-FLAG-BAP1-F170I was generated by site-directed mutagenesis to create the amino acid substitution phenylalanine to isoleucine at codon 170.
The Tnnt2 cDNA was amplified from murine cardiac RNA by reverse transcription-polymerase chain reaction (RT-PCR) (Qiagen) with primers F3, 5'- andCCTGTGTCGACTCCCTGTTCAGAGGGAGAGCCGAGAG-3', and R4, 5'- andCAGGAGthenCTTTGGGTGCCAAGGAGGACCCAGAGC-3', and then subjected to PCR mutagenesis to delete nucleotides AAG at positions 628 630, encoding the lysine deletion at codon 210 (K210Δ).
Derivative plasmids containing mutations of the ATGs at codon 1 and codon 26 were generated by site-directed mutagenesis.
We therefore introduced base exchanges at codon 374 of the murine Cox-2 cDNA by site-directed mutagenesis and stably expressed the resulting Cox-2H374E Cox-2H374F374F mutants in E1A-MEF.
