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  • Title: Ethyl methane sulfonate- and bleomycin-generated deletion mutations at HPRT locus in xeroderma pigmentosum complementation group D fibroblasts.
    Author: Wood CM, Moses RE.
    Journal: Somat Cell Mol Genet; 1989 Jul; 15(4):345-57. PubMed ID: 2474861.
    Abstract:
    Immortalized fibroblasts from a male patient with xeroderma pigmentosum from complementation group D (XP-D) were treated with either ethyl methane sulfonate (EMS) or bleomycin (BLM) to obtain mutations in hypoxanthine phosphoribosyltransferase (HPRT) activity. The aneuploid parental cell line, MH3-XPD, was found to have a single copy of the HPRT gene, indicating that this cell line remained physically hemizygous for this locus during the transformation process. Subcloning of 6-thioguanine-resistant (6TG') isolates resulted in clones without detectable HPRT activity. Continued maintenance in elevated concentrations of 6TG (30-60 muM) produced cell populations with negligible growth in counterselection medium. No HPRT-deficient clones arose from unmutagenized cell cultures. Molecular analysis of the HPRT mutations in five clones with undetectable HPRT activity showed that four had large deletions. Two bleomycin-generated isolates were both found to have an approximately 28-kb intragenic deletion beginning with the first intron near exon 1 and ending within the fourth intron near exon 4. Messenger RNA from these clones was truncated by approximately 370 nucleotides. Our findings indicate that these two clones originated from the same mutational event within a founder cell. The three EMS-induced mutants fell into two classes: a putative point mutation or small deletion and two complete gene deletions.
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