Single amino acid substitution defines a naturally occurring genetic variant of human thymidylate synthase.
Previously, we identified an altered structural form of thymidylate synthase (TS) in a human colonic tumor cell line. This form, which is encoded by a variant structural gene, renders cells relatively resistant to 5-fluoro-2'-deoxyuridine as a result of the reduced affinity of the enzyme for the active metabolite 5-fluoro-2'-deoxyuridylic acid. We have isolated a cDNA clone specific to the altered TS and have determined its sequence. Two point mutations distinguish the normal from the altered TS mRNAs. One, a (A----G) change, is located within the 3'-untranslated region; the other, a T----C change within the amino acid-coding region, predicts replacement of tyrosine by histidine at residue 33 of the polypeptide. This sequence change was confirmed by direct analysis of cDNA amplified by the polymerase chain reaction and was further verified using allele-specific oligonucleotides as probes in Northern blots. These results, along with studies by other laboratories showing Tyr33 to be evolutionarily conserved, suggest that this residue plays an important role in TS function.
Barbour
K W
KW
Department of Biological Sciences, University of South Carolina, Columbia 29208.
Berger
S H
SH
Berger
F G
FG
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D004247:DNA / Q000302:isolation & purification; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics*; D006639:Histidine / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D013940:Thymidylate Synthase / Q000032:analysis / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000032:analysis*
D004273:DNA, Neoplasm; D015345:Oligonucleotide Probes; D014443:Tyrosine; D006639:Histidine; D004247:DNA; D013940:Thymidylate Synthase