Yonsei Med J.  1991 Sep;32(3):207-214. 10.3349/ymj.1991.32.3.207.

Identification of mutagenic site of c-H-ras oncogene damaged by N-acetoxyacetylaminofluorene(AAAF)

• Oh SH
• Kim KH
• Lee SE
• Yeh BI

• KMID: 1729587
• DOI: http://doi.org/10.3349/ymj.1991.32.3.207

Abstract

A molecularly cloned human cellular H-ras (c-H-ras) oncogene(pbc N1 plasmid) was treated with N-acetoxyacetylaminofluorene (AAAF) in vitro and subcloned into E.coli. This was done to identify the mutational changes at specific codons of the gene. Guanine nucleotides were identified as the major AAAF binding site of the DNA adduct formed. Base changes in codons 12 and 61 were determined by the analysis of restriction fragment length polymorphism (RFLP) and site specific oligonucleotide hybridization. RFLP was observed due to the loss of the Hpall recognition site at codon 11 and 12 of AAAF-treated c-H-ras gene. Hybridization of AAAF treated c-H-ras with 32P-labeled oligonucleotide probes for the mutant alleles of codon 61 showed no substitutions at codon 61. From these results, it is assumed that AAAF treatment in vitro caused mutation at codon 12 but not at codon 61 of the c-H-ras oncogene and that codon 12 is the primary target of mutation by AAAF

Keyword

c-H-ras oncogene; AAAF; mutation

MeSH Terms

Acetoxyacetylaminofluorene/*pharmacology
Chromatography, Thin Layer
Codon
*DNA Damage
Electrophoresis, Agar Gel
Genes, ras/*drug effects/genetics
Human
Mutagenesis, Site-Directed
Oligonucleotide Probes
Plasmids/drug effects/genetics
Polymorphism, Restriction Fragment Length