Loss of heterozygosity in mammalian cell mutagenesis: molecular analysis of spontaneous mutations at the aprt locus in CHO cells.
Loss of heterozygosity at previously heterozygous loci may occur by one of several possible mechanisms and account for a large fraction of all mutations occurring at such loci. In order to investigate loss of heterozygosity events, we have chosen the aprt locus of Chinese hamster ovary (CHO) cells as our model since it is readily available in either heterozygous or hemizygous form. Cloning and sequencing of the two heterozygous aprt alleles from the CHO derivative D423 identified a single polymorphic site, which does not create a restriction fragment length polymorphism. In order to evaluate the loss of heterozygosity events at this locus, we devised a method that creates an artificial restriction fragment length polymorphism in one of these two alleles as a direct consequence of enzymatic amplification. Restriction enzyme digestion of the amplified sequences can then conveniently identify the genotype of the DNA sample. This same methodology also provides for the selective cloning of only one allele of a heterozygous pair into a plasmid vector for subsequent DNA sequence analysis, and can be easily adapted to other situations requiring the analysis of single base changes at a particular position within known sequences. Using this technique, we have determined that 16/37 (43%) spontaneous APRT- mutants had undergone a loss of heterozygosity event.
Ward
M A
MA
Department of Medical Genetics, University of Toronto, Ontario, Canada.
Yu
M
M
Glickman
B W
BW
Grosovsky
A J
AJ
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics*; D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006579:Heterozygote; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010053:Ovary; D010430:Pentosyltransferases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
D015345:Oligonucleotide Probes; D010430:Pentosyltransferases; D000228:Adenine Phosphoribosyltransferase