Experiments were designed to test the assumption that the conserved residues (CR) of a protein are essential for its function. Procedures were developed for the efficient use of the polymerase chain reaction (PCR) to introduce alterations into codons for CRs in a region of the vaccinia virus DNA polymerase. These alterations are linked to a mutation in an adjacent site which confers resistance to aphidicolin (AP), a drug which is a competitive inhibitor for cytidine deoxyribonucleotide triphosphate (dCTP). A single priming oligonucleotide with an alteration of a codon for a specific CR and the mutation which confers AP resistance (APr) was used in the PCR to produce an altered DNA which was introduced into virus infected cells by marker transfer (MT). An extract of these cells was diluted and altered virus was detected by plaque formation in AP medium. The most significant substitution to date was the replacement of a pair of amino acids, tyrosine and alanine, by tryptophan and threonine, in the same sequence. Vaccinia virus with these alterations grew at the normal rate and produced a normal yield of plaques. No plaques were detected after marker transfer when more """"""""drastic"""""""" substitutions of highly conserved residues, such as substituting aspartic acid for tyrosine, were attempted. This result indicates that within the limits of the MT reaction such substitutions are not allowed. Controls for the MT reaction included a standard substitution which utilizes an oligonucleotide that inserts a Bg1I restriction site into the region with the CRs. In addition, every priming oligonucleotide which contained an alteration of a CR also contained the alterations to introduce the Bg1 site as well as the APr mutation. PCR methods were developed to amplify small amounts of virus DNA from APr plaques to check the sequence of the DNA for the desired alterations.
