Genetic analysis of a vital mammalian housekeeping locus using CHO cells that express a transfected mutant allele.
We describe a novel approach for the isolation of null mutations in a vital Chinese hamster ovary (CHO) cell housekeeping gene. Our experimental strategy required introduction of an expressible DNA clone encoding a recessive emetine-resistance allele of ribosomal protein S14 into wild-type CHO cells. Transgene heterozygote (TGH) cell lines, which harbor multiple emetine-resistance S14 transgenes, survive mutations that inactivate the CHO RPS14 locus by virtue of the transgenes' biological function. Null mutations in RPS14 yield TGH clones that display the transgene's drug-resistance phenotype. A large collection of emetine-resistant clones was isolated from one TGH cell line and shown to consist of three types of S14 mutations: (1) nonsense null mutations in the RPS14 protein coding sequence; (2) missense null mutations that affect S14 amino acid residues that have been conserved stringently during eukaryotic evolution; and (3) a recurrent missense mutation that results in a new, functional RPS14 emetine-resistance allele.
Diaz
J J
JJ
Division of Biology, Kansas State University, Manhattan 66506.
Rhoads
D D
DD
Roufa
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012269:Ribosomal Proteins / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D012333:RNA, Messenger; D012269:Ribosomal Proteins; C039747:ribosomal protein S14