A system for assaying homologous recombination at the endogenous human thymidine kinase gene.
A system for assaying human interchromosomal recombination in vitro was developed, using a cell line containing two different mutant thymidine kinase genes (TK) on chromosomes 17. Heteroalleles were generated in the TK+/+ parent B-lymphoblast cell line WIL-2 by repeated exposure to the alkylating nitrogen mustard ICR-191, which preferentially causes +1 or -1 frameshifts. Resulting TK-/- mutants were selected in medium containing the toxic thymidine analog trifluorothymidine. Mutations were characterized by exon-specific polymerase chain reaction amplification and direct sequencing. In two lines, heterozygous frameshifts were located in exons 4 and 7 of the TK gene separated by approximately 8 kilobases. These lines undergo spontaneous reversion to TK+ at a frequency of less than 10(-7), and revertants can be selected in cytidine/hypoxanthine/aminopterin/thymidine medium. The nature and location of these heteroallelic mutations make large deletions, rearrangements, nondisjunction, and reduplication unlikely mechanisms for reversion to TK+. The mode of reversion to TK+ was specifically assessed by DNA sequencing, use of single-strand conformation polymorphisms, and analysis of various restriction fragment length polymorphisms (RFLPs) linked to the TK gene on chromosome 17. Our data suggest that a proportion of revertants has undergone recombination and gene conversion at the TK locus, with concomitant loss of frameshifts and allele loss at linked RFLPs. Models are presented for the origin of two recombinants.
Department of Cancer Biology, Harvard School of Public Health, Boston, MA 02115.
M B
MB
Benjamin
H
H
Potter
D W
DW
Yandell
J B
JB
Little
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D005091:Exons; D005796:Genes; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D011995:Recombination, Genetic; D013937:Thymidine Kinase / Q000235:genetics*
D013937:Thymidine Kinase