Molecular analysis of 5-azacytidine-induced variants in mammalian cells.
5-azacytidine induces 6-thioguanine resistance in AS52 cells. To characterize these resistant clones, we isolated 148 of them from 50 independently treated flasks. Less than nine (6%) of the 148 variants were spontaneous. PCR amplification of the DNA primers flanking the gpt gene produced no product in 15 clones (10%). Of the 133 remaining clones, 52 showed sequence alterations in the gpt structural gene. Of these 52, 34 (65%) were GC-->CG transversions. Only seven were located in CpG sequences. Thus, methyltransferase complexes are not major contributors to 5-azacytidine-induced point mutations in AS52 cells. The remaining 81 clones had no sequence alterations within the coding region of the gpt gene. Southern blot analysis of a sample of these variants (37/81) indicated that the 6-thioguanine-resistant phenotype was not due to local rearrangements or deletions (resolution 50 bp). Sequence analysis of the early promoter region of another sample of these variants (24/81) indicated that lesions in the promoter could not be responsible for the 6-thioguanine resistance observed. Thus, a majority of these variants were formed via a mechanism other than small genomic rearrangements, point mutations or deletions of the gpt structural gene or its promoter. Neither the mechanisms leading to these variants nor the biological and morphological consequences of these variants are known.
Kelecsényi
Z
Z
Cancer Genetics Group, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Spencer
D L
DL
Caspary
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D001374:Azacitidine / Q000633:toxicity*; D001483:Base Sequence; D015139:Blotting, Southern; D016466:CHO Cells; D018899:CpG Islands; D006224:Cricetinae; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008969:Molecular Sequence Data; D009153:Mutagens / Q000633:toxicity*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D009153:Mutagens; D013762:Tetrahydrofolate Dehydrogenase; D007041:Hypoxanthine Phosphoribosyltransferase; D001374:Azacitidine