Construction of a specific amber codon in the simian virus 40 T-antigen gene by site-directed mutagenesis

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Abstract
The site-directed bisulfite mutagenesis technique was used to construct a specific mutation, am404, at nucleotide position 3124 in the SV-40 genome. The mutation was contained within a PstI restriction site (map position 0.27) and prevented cleavage by PstI at that position. Nucleotide sequence analysis of the mutagenized region indicated that only a single base pair change had occurred: a guanosine.cntdot.cytosine .fwdarw. adenine.cntdot.thymidine transition. Comparison of the nucleotide sequence of am404 with the known DNA sequence of SV-40 indicated that the mutation in am404 resulted in the conversion of a glutamine codon to an amber codon. The am404 could not replicate autonomously when transferred into [African green] monkey [kidney] cells (BSC-40) but did replicate when it was co-transfected with the late deletion helper virus dl1007. On the basis of its position in the T-antigen, gene am404 should produce a T-antigen 24% shorter than the wild-type protein.