SEQUENCE OF THE RAT HYPOXANTHINE-GUANINE PHOSPHORIBOSYL TRANSFERASE (HPRT) TRANSCRIPTIONAL PROMOTER REGION IN WILD-TYPE AND MUTANT RAT-LIVER EPITHELIAL-CELL LINES

The hypoxanthine guanine phosphoribosyltransferase (HPRT) gene is muta ted by a variety of genotoxic agents in adult rat liver (ARL) epitheli al cell lines. By polymerase chain reaction (PCR) amplification and DN A sequencing of rat ARL cell HPRT gene sequences with mouse- and rat-s pecific oligonucleotides, a large portion of the rat HPRT transcriptio nal promoter region was sequenced. This region exhibits approximately 60% homology with the corresponding mouse sequence, contains a similar G/C-rich region at its 3' end, and contains a similar series of 6-nuc leotide (nt) GGGCGG repeats. To determine if this region is a target f or mutation by different genotoxins, HPRT-deficient ARL mutants induce d by 2-acetylaminofluorene (AAF), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or 7,12-dimethyl-benz[a]anthracene (DMBA) were isolated and s tudied. A 1003-nt fragment of predominantly HPRT regulatory sequences was amplified by PCR using purified genomic DNA from 17 independent mu tants and sequenced directly. None of the 17 mutants examined exhibite d any alterations in the transcriptional regulatory region or the 5' u ntranslated region of HPRT exon 1 after direct sequencing analysis of PCR products. In addition, none of the 2-AAF-induced mutants exhibited differences in in vitro transcription rates as determined by nuclear run-on analysis. These data suggest that regulatory sequences of the H PRT gene are not a primary target for mutation by the genotoxins studi ed.