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 thy A 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 flex neri Y was identical with that of the thyA mutant except that the resi due T at position 345 was replaced by residue A in the thyA mutant. Th is change would cause a predicted amino acid substitution of leucine a t 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.