Low hprt mRNA levels and multiple hprt mRNA species in 6-thioguanine-resistant Chinese hamster cell mutants possessing nonsense mutations.
Previous studies suggested that many Chinese hamster ovary (CHO) cell hprt mutants having point mutations in the protein coding region also have low steady-state hprt mRNA concentrations. In addition, polymerase-chain reaction (PCR) amplification of hprt cDNA synthesized from some of these mutants results in multiple products containing deleted exons indicating that these mutants possess multiple species of hprt mRNA. In this study, we have used northern blot analysis to quantify the concentrations of hprt mRNA in 86 mutants known to possess point mutations leading to either missense or nonsense mutations. 28 of 35 nonsense mutants (80%), but only 7 of 51 missense mutants (14%), had < 50% of the hprt mRNA concentration found in parental CHO cells. Furthermore, all the nonsense mutants with premature termination codons in the internal exons of the gene (i.e., exons 3, 4, 5, 6 and 7) showed a significant reduction (averages < 16% of parental) in the steady-state levels of hprt mRNA, while nonsense mutants with termination codons situated in the extreme 5' and 3' regions of the gene had near parental hprt mRNA levels. In the same collection of mutants, the proportion of mutants producing multiple cDNA PCR products was much greater (18/35) for mutants having nonsense mutations than for mutants with missense mutations (2/51). All nonsense mutants with mutations in exons 3, 4 and 5 produced multiple species, while all those with mutations in exons 7, 8 and 9 produced a single PCR product. These results suggest that sequence changes in mammalian genes that affect protein chain length can also affect mRNA concentration and the splicing of pre-mRNA molecules.
Manjanatha
M G
MG
National Center for Toxicological Research, Division of Genetic Toxicology, Jefferson, AR 72079.
Lindsey
L A
LA
Mittelstaedt
R A
RA
Heflich
R H
RH
D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D006224:Cricetinae; D004351:Drug Resistance; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D013866:Thioguanine / Q000494:pharmacology
D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine