Amsacrine-induced mutations in AS52 cells

✍ Scribed by Lynnette R. Ferguson; Pamela M. Turner; Daniel W. Hart; Kenneth R. Tindall

• Book ID: 101265335
• Publisher: John Wiley and Sons
• Year: 1998
• Tongue: English
• Weight: 221 KB
• Volume: 32
• Category: Article
• ISSN: 0893-6692
• DOI: 10.1002/(sici)1098-2280(1998)32:1<47::aid-em6>3.0.co;2-8

✦ Synopsis

Amsacrine is an acridine-derived inhibitor of topo-quences was found in 1 (2%) of the mutants, and isomerase II that intercalates into DNA. We per-7 (14%) of the mutant clones had altered PCR patformed a detailed molecular analysis of 6-thiogua-terns, suggesting complex deletions or rearrangenine (6-TG)-resistant mutant colonies arising in ments. The remaining 42 (84%) mutants had a wild-AS52 cells following Amsacrine treatment. AS52 type PCR profile. Of these, 21 mutants were further cells carry a single copy of the bacterial gpt gene, analysed by Southern blotting. Interestingly, Southfunctionally expressed using the SV40 early pro-ern blotting revealed genomic deletions/rearrangemoter and stably integrated into the Chinese ham-ments in 12 of 21 mutants with a wild-type PCR ster ovary genome. A 1-hr treatment with 0.1 to 0.5 profile. These deletions/rearrangements were fur-mM Amsacrine was both cytotoxic and mutagenic, ther shown to affect gpt gene expression. The reresulting in an average mutant frequency (MF) of maining nine mutants with a wild-type PCR profile 143 1 10 06 at 0.5 mM. Fifty independent 6-TG-were sequenced. Four of these mutants had mutaresistant colonies were isolated for further study. tions in the gpt structural gene. Overall, genomic These clones were initially characterised by PCR deletions/rearrangements were observed in 12/21 to estimate the relative proportion of putative point independent mutants subjected to PCR and Southmutants and deletions or rearrangements; then a ern blotting. Thus, deletions/rearrangements were subset of mutants was further characterised by the most common mutation observed following Am-Southern blotting, Northern blotting, and DNA se-sacrine treatment of AS52 cells.