Analysis of mutations in alleles of thefur gene from an endoprotease-deficient chinese hamster ovary cell strain

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Abstract
RPE. 40 mutant cells differ from wild-type Chinese hamster ovary (CHO-K1) cells in their increased resistance toPseudomonas exotoxin A and their inability to process the insulin proreceptor and certain viral envelope proproteins. Northern analysis revealed that RPE. 40 cells maintained a substantially lower steady-state level of 4.0 kbfur mRNA than did CHO-K1 cells. Analysis offur cDNAs showed that RPE. 40 cells were diploid at thefur locus, and RPE. 40 cells had a Cys (TGC) to Tyr (TAC) mutation in codon 196 of one allele (allele I). Approximately 25–30% of the CHO-K1 cells were also heterozygous (Tyr/Cys) at codon 196, and pre-mRNAs transcribed from the second allele (allele II) in RPE. 40 cells were defectively spliced. All other pre-mRNAs were correctly spliced. Rapid turnover of defectively spliced transcripts may account for the reduced steady-state level offur mRNA observed in RPE. 40 cells. Our results provide a mechanistic basis for the endoprotease-deficient phenotype of RPE. 40 cells.