MOLECULAR-CLONING OF THE WILD-TYPE AND MUTANT THYA GENE FROM SHIGELLA-FLEXNERI-Y

The thyA gene which codes for thymidylate synthase has been cloned and sequenced from the wild-type Shigella flexneri Y strain SH4 and a thyA mutant TSF21 after amplifying the gene by polymerase chain reaction (PCR). The nucleotide sequence revealed 98% homology to the E. coli K-12 thyA gene. The sequence of the wild-type thyA gene of Shigella flexneri Y was identical with that of the thyA mutant except that the residue T at position 345 was replaced by residue A in the thyA mutant. This change would cause a predicted amino acid substitution of leucine at position 44 in the polypeptide product of the wild type by glutamine in the mutant. Thus, Leu(44) may be critical in enzymatic activity of the thyA gene product thymidylate synthase.