Isolation of an APRT heterozygote from TK6 human lymphoblasts: predominance of multi-locus loss of heterozygosity among spontaneous APRT-mutants.
The TK6 human B lymphoblastoid cell line contains two easily and widely used selectable markers: the X-linked, hemizygous hprt locus, and the heterozygous tk locus on chromosome 17q. In this study, rare APRT heterozygotes were directly isolated from the TK6 population by clonal selection in cell culture medium supplemented with 5 micrograms/ml of 8-azaadenine. One of nine isolated heterozygotes, AZH1, was characterized extensively. APRT- mutants can be recovered from AZH1 at a mutation rate of 1.5 x 10(-7), similar to rates previously determined for the selection of TK- and HPRT- mutants from TK6. A unique sequence alteration was identified in the non-functional aprt allele at position 1930. A G:C to A:T transition at this site alters the canonical AG splice acceptor dinucleotide in exon 3, and also results in the destruction of a Stul recognition sequence. This polymorphism was used to analyze loss of heterozygosity in a set of 32 spontaneous APRT- mutants by restriction analysis following PCR amplification. Analysis of flanking microsatellite dinucleotide polymorphisms demonstrated that LOH occurring in spontaneous APRT- mutants is nearly always a multi-locus event extending at least 7.5 cM along chromosome 16q. This pattern of LOH among APRT- mutants differs from extensive LOH in spontaneous, normal-growth TK- mutants derived from TK6 cells (p < 0.0001), and suggests that cis-acting factors may be equally important in shaping the mutational spectrum as trans-acting factors such as cellular apoptotic capacity.
Pongsaensook
P
P
Environmental Toxicology Graduate Program, University of California, Riverside 92521, USA.
Smith
L E
LE
Grosovsky
A J
AJ
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000228:Adenine Phosphoribosyltransferase / Q000235:genetics*; D001402:B-Lymphocytes / Q000201:enzymology*; D002999:Clone Cells; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006579:Heterozygote; D006801:Humans; D018895:Microsatellite Repeats; D009154:Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured
D000228:Adenine Phosphoribosyltransferase; C028868:AGGCCT-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D000225:Adenine; C006654:8-azaadenine