A mutation in the vaccinia virus DNA polymerase gene which changes amino acid residue 645 in the polymerase from leucine to methionine results in a vaccinia virus mutant which is resistant to aphidicolin (AP). Residue 645 is in a region of conserved amino acids in the carboxyl portion of the polymerase. I use site directed mutagenesis to change the conserved amino acids near residue 645 to determine how essential these amino acids are for enzyme activity. The strategy is to use the marker transfer procedure to introduce linked alterations one of which alters a codon for a conserved residue while the other confers AP resistance. Virus plaque formation in the presence of AP indicates that the polymerase can function with the new amino acid since recombination between the linked chances should be minimal. To test the method, I used an altered DNA with a neutral change from valine to alanine, at residue 645. Marker transfer produced AP-resistant plaques whose progeny contained DNA with the codon for alanine at the proper position. However, when I tried to introduce drastic changes, such as replacing asparagine (residue 640) with aspartic acid, or serine (residue 641) with proline, or tyrosine (residue 643) with aspartic acid, the attempts failed. Although AP-resistant plaques were formed after marker transfer, the progeny from the plaques contained only the wild-type codon at the proper position. The reason for this result is not obvious, so the experiment will be improved and repeated. One improvement removes contaminating DNA in the polymerase chain reaction (PCR) which amplifies the altered DNA. Another advance involves the ability to extract DNA from the virus so that the DNA is suitable for amplification without undue manipulation. Finally, a method was developed which allows rapid sequencing of DNA from the PCR.
