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Characterization of hprt splicing mutations induced by the ultimate carcinogenic metabolite of benzo[a]pyrene in Chinese hamster V-79 cells.
Cancer Res. 1995 Apr 01; 55(7):1550-8.CR

Abstract

The molecular basis for putative aberrant splicing of hypoxanthine (guanine) phosphoribosyltransferase (hprt) pre-mRNA in Chinese hamster V-79 cells was determined for 75 independent (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene [(+)-BPDE]-induced and 6 spontaneous 8-azaguanine-resistant mutant clones that had exon deletions in their hprt cDNA. Genomic DNA fragments corresponding to the missing exons and their flanking intron regions were amplified by PCR and sequenced. The results indicated that each of these mutants generated a normal-sized PCR product and resulted from aberrant splicing. For (+)-BPDE-induced aberrant splicing mutants, 81% (61 of 75 clones) had base substitution mutations, 5% (4 of 75 clones) had a single base deletion, and 13% (10 of 75 clones) lacked a detectable mutation in the skipped exon, its flanking intron sequences, or in the upstream donor site of the preceding intron. All mutations at a splice donor site resulted in skipping of the entire upstream neighboring exon, whereas alterations at a splice acceptor site caused skipping of the downstream neighboring exon or activation of a cryptic acceptor site in the downstream exon. Fifty-nine % of the splicing mutants had a mutation occurring at the splice site consensus sequence in the intron, and 28% of the splicing mutants had mutations within exon sequences. Among 21 aberrant splicing mutant clones with a mutation inside an exon sequence, seven were in exon 2, two were in exon 3, and twelve were in exon 4. Evidence is presented that a stemloop structure sequesters the splice donor site of exon 2 in pre-mRNA and plays a role in exon 2 skipping. Mutant clones with mutations stabilizing the proposed stemloop structure inhibited the use of the normal exon 2 splice site which resulted in exon 2 skipping in the hprt mRNA. These mutant clones expressed a mixed population of mRNAs, and both normal-sized and truncated mRNA were formed. Similar to our earlier finding that treatment of V-79 cells with (+)-BPDE resulted in a dose-dependent mutation profile within the coding region of the hprt gene, we also observed the presence of dose-dependence in the profile of (+)-BPDE-induced base substitutions in aberrant splicing mutants. As the dose of (+)-BPDE was decreased, the proportion of base substitution mutations at AT base pairs that affected RNA splicing was increased.

Authors+Show Affiliations

Department of Chemical Biology and Pharmacognosy, College of Pharmacy, Rutgers, State University of New Jersey, Piscataway 08854.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

7882364

Citation

Hennig, E E., et al. "Characterization of Hprt Splicing Mutations Induced By the Ultimate Carcinogenic Metabolite of Benzo[a]pyrene in Chinese Hamster V-79 Cells." Cancer Research, vol. 55, no. 7, 1995, pp. 1550-8.
Hennig EE, Conney AH, Wei SJ. Characterization of hprt splicing mutations induced by the ultimate carcinogenic metabolite of benzo[a]pyrene in Chinese hamster V-79 cells. Cancer Res. 1995;55(7):1550-8.
Hennig, E. E., Conney, A. H., & Wei, S. J. (1995). Characterization of hprt splicing mutations induced by the ultimate carcinogenic metabolite of benzo[a]pyrene in Chinese hamster V-79 cells. Cancer Research, 55(7), 1550-8.
Hennig EE, Conney AH, Wei SJ. Characterization of Hprt Splicing Mutations Induced By the Ultimate Carcinogenic Metabolite of Benzo[a]pyrene in Chinese Hamster V-79 Cells. Cancer Res. 1995 Apr 1;55(7):1550-8. PubMed PMID: 7882364.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization of hprt splicing mutations induced by the ultimate carcinogenic metabolite of benzo[a]pyrene in Chinese hamster V-79 cells. AU - Hennig,E E, AU - Conney,A H, AU - Wei,S J, PY - 1995/4/1/pubmed PY - 1995/4/1/medline PY - 1995/4/1/entrez SP - 1550 EP - 8 JF - Cancer research JO - Cancer Res VL - 55 IS - 7 N2 - The molecular basis for putative aberrant splicing of hypoxanthine (guanine) phosphoribosyltransferase (hprt) pre-mRNA in Chinese hamster V-79 cells was determined for 75 independent (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene [(+)-BPDE]-induced and 6 spontaneous 8-azaguanine-resistant mutant clones that had exon deletions in their hprt cDNA. Genomic DNA fragments corresponding to the missing exons and their flanking intron regions were amplified by PCR and sequenced. The results indicated that each of these mutants generated a normal-sized PCR product and resulted from aberrant splicing. For (+)-BPDE-induced aberrant splicing mutants, 81% (61 of 75 clones) had base substitution mutations, 5% (4 of 75 clones) had a single base deletion, and 13% (10 of 75 clones) lacked a detectable mutation in the skipped exon, its flanking intron sequences, or in the upstream donor site of the preceding intron. All mutations at a splice donor site resulted in skipping of the entire upstream neighboring exon, whereas alterations at a splice acceptor site caused skipping of the downstream neighboring exon or activation of a cryptic acceptor site in the downstream exon. Fifty-nine % of the splicing mutants had a mutation occurring at the splice site consensus sequence in the intron, and 28% of the splicing mutants had mutations within exon sequences. Among 21 aberrant splicing mutant clones with a mutation inside an exon sequence, seven were in exon 2, two were in exon 3, and twelve were in exon 4. Evidence is presented that a stemloop structure sequesters the splice donor site of exon 2 in pre-mRNA and plays a role in exon 2 skipping. Mutant clones with mutations stabilizing the proposed stemloop structure inhibited the use of the normal exon 2 splice site which resulted in exon 2 skipping in the hprt mRNA. These mutant clones expressed a mixed population of mRNAs, and both normal-sized and truncated mRNA were formed. Similar to our earlier finding that treatment of V-79 cells with (+)-BPDE resulted in a dose-dependent mutation profile within the coding region of the hprt gene, we also observed the presence of dose-dependence in the profile of (+)-BPDE-induced base substitutions in aberrant splicing mutants. As the dose of (+)-BPDE was decreased, the proportion of base substitution mutations at AT base pairs that affected RNA splicing was increased. SN - 0008-5472 UR - https://www.unboundmedicine.com/medline/citation/7882364/Characterization_of_hprt_splicing_mutations_induced_by_the_ultimate_carcinogenic_metabolite_of_benzo[a]pyrene_in_Chinese_hamster_V_79_cells_ L2 - http://cancerres.aacrjournals.org/cgi/pmidlookup?view=long&pmid=7882364 DB - PRIME DP - Unbound Medicine ER -