title: The association of nonsense mutation with exon-skipping in hprt mRNA of Chinese hamster ovary cells results from an artifact of RT-PCR.
abstract: RT-PCR of RNA from CHO cells with nonsense mutations in the hprt gene frequently detects minor hprt mRNA species lacking one or more exons. Many nonsense mutants also contain greatly reduced concentrations of the major, normally spliced hprt mRNA. In this study, we examined the hypothesis that exon-deleted mRNAs are normal constituents of CHO cells, but are not detected in wild-type parental cells and most missense mutants because their amplification is suppressed by relatively high concentrations of normally spliced hprt mRNA. A protocol designed to specifically detect exon-deleted mRNAs was conducted using RNA from parental cells and identified all the exon-deleted species typical of nonsense mutants. Quantitative analysis of parental cell RNA measured these exon-deleted mRNAs at < or = 0.7% of the abundance of the full-sized species. Nonsense and missense mutants had comparable amounts of exon-deleted mRNAs, which varied both above and below parental concentrations. The relative concentrations of particular exon-deleted species could be explained by the location of nonsense mutations remaining in the mRNA or by structural effects of mutations on splicing. Exon-deleted mRNAs were detected by RT-PCR when the concentration of the most abundant exon-deleted species was > or = 2% of the full-length mRNA. This occurred for mutants with nonsense mutations in internal exons. RT-PCR conditions were shown to suppress the amplification of exon-deleted species 40-fold when full-length mRNA was abundant, which occurred for parental lines and missense mutants. Our results verify that RT-PCR conditions can produce an artifactual association between nonsense mutation and exon-skipping when minor, exon-deleted mRNA is relatively enriched.
authors.affiliation: Division of Genetic Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079-9502, USA. cvalentine@nctr.fda.gov
authors.forename: C R
authors.initials: CR
authors.lastname: Valentine
authors.forename: R H
authors.initials: RH
authors.lastname: Heflich
mesh_terms: D000818:Animals; D016477:Artifacts; D016466:CHO Cells; D018389:Codon, Nonsense; D006224:Cricetinae; D005091:Exons / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D014158:Transcription, Genetic
chemical_list: D018389:Codon, Nonsense; D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase