Analysis of mutations in alleles of the fur gene from an endoprotease-deficient Chinese hamster ovary cell strain.
RPE.40 mutant cells differ from wild-type Chinese hamster ovary (CHO-K1) cells in their increased resistance to Pseudomonas 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 kb fur mRNA than did CHO-K1 cells. Analysis of fur cDNAs showed that RPE.40 cells were diploid at the fur 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 of fur mRNA observed in RPE.40 cells. Our results provide a mechanistic basis for the endoprotease-deficient phenotype of RPE.40 cells.
Spence
M J
MJ
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, Burlington, Vermont 05405, USA.
Sucic
J F
JF
Foley
B T
BT
Moehring
T J
TJ
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000302:isolation & purification; D005260:Female; D045683:Furin; D008969:Molecular Sequence Data; D017354:Point Mutation; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment; D013381:Subtilisins / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D013381:Subtilisins; D045683:Furin