Molecular analysis of mutations induced by 4'-hydroxymethyl-4,5',8-trimethylpsoralen and UVA in the mouse HPRT gene.
The effects of the reaction photosensitized by 4'-hydroxymethyl-4,5'-8-trimethylpsoralen (HMT) on a mouse lymphoma cell line have been examined. Using the hypoxanthine phosphoribosyltransferase (HPRT) locus as target gene, a mutagenic effect of the photoreaction can be detected concomitantly with a loss of cell viability. Isolation of HPRT deficient clones has permitted a molecular characterization of the mutational pattern induced by the photosensitization reaction mediated by HMT. Southern blotting analysis demonstrated that the HPRT deficiency could not be correlated with gene deletions larger than 300 bp. Using polymerase chain reaction on both DNA and cDNA, amplification products have been cloned into M13mp18 and sequenced. Base transversions targeted on thymine residues have been located in exon 2, 3, 8 and 9 together with spontaneous frameshift mutations occurring in a run of guanine residues in exon 3. HPRT deficiencies owing to mutations arising in the HPRT promoter region have also been observed. Dot and Northern blot analysis revealed that the photoreaction could lead to either a reduced level of gene transcription or to a complete absence of HPRT m-RNA. Using polymerase chain reaction (PCR) amplification and agarose gel electrophoresis, deletions in the HPRT promoter have been observed and correlated to deficient enzyme expression.
Piette
J
J
Laboratory of Microbiology, Institute of Pathology B23, University of Liège, Belgium.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004273:DNA, Neoplasm / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005796:Genes / Q000187:drug effects* / Q000528:radiation effects; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008223:Lymphoma; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014307:Trioxsalen / Q000031:analogs & derivatives* / Q000494:pharmacology; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm; D009838:Oligodeoxyribonucleotides; C016507:hydroxymethyltrioxsalen; D007041:Hypoxanthine Phosphoribosyltransferase; D014307:Trioxsalen