Construction of a specific amber codon in the simian virus 40 T-antigen gene by site-directed mutagenesis.
The site-directed bisulfite mutagenesis technique has been used to construct a specific mutation, am404, at nucleotide position 3124 in the simian virus 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 x cytosine leads to adenine x thymine transition. Comparison of the nucleotide sequence of am404 with the known DNA sequence of simian virus 40 indicted that the mutation in am404 resulted in the conversion of a glutamine codon to an amber codon. am404 could not replicate autonomously when transfected into monkey cells (BSC-40) but did replicate when it was cotransfected 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.
D000951:Antigens, Neoplasm / Q000235:genetics*; D000956:Antigens, Viral / Q000235:genetics*; D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D004279:DNA, Viral / Q000096:biosynthesis; D005814:Genes, Viral; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics*; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection
D000951:Antigens, Neoplasm; D000956:Antigens, Viral; D003062:Codon; D004279:DNA, Viral; D012333:RNA, Messenger