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+/I 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 exonspecific 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 -8 kilobases.These lines undergo spontaneous reversion to TKV at a frequency of <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 TKV 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.Molecular genetic studies suggest that somatic recombination and gene conversion may play an important role in the generation of certain human diseases. Of particular interest is the reduction to homozygosity of mutations at tumorsuppressor loci as the cause of a proportion of retinoblastomas, osteosarcomas (1-3), Wilm tumors (4-6), astrocytomas (7), and meningiomas and acoustic neuromas (8,9). Previous analyses of recombination in cultured mammalian cells have used extrachromosomal plasmid-based systems or integrated markers in tandem array (10-17), but these may not be appropriate models for recombinational events between autosomal genes in their native chromosomal environment. The identification of interchromosomal homologous recombination in mammalian cells in vitro has been largely confined to detecting post-S-phase recombination and the reduction to homozygosity of large chromosomal regions where the result is loss of a dominant (usually wild-type) allele or the identification ofrecombination-induced cytogenetic changes (18-21).We describe a system for the analysis of recombination between alleles of the endogenous human thymidine kinase gene (TK) on chromosome 17. The assay detects recombination events that are initiating within or migrate through this gene. TK-deficient (TK-/-) lymphoblasts were generated from a near-diploid TK+'/ line by repeated exposure to the frameshift mutagen . Frameshift mutations in three mutant lines were located and characterized by TK exon-specific polymerase chain reaction (PCR) amplification...

We have developed a human lymphoblast cell line for the study of interchromosomal homologous recombination at the endogenous thymidine kinase (tk) gene on chromosome 17 (M. B. Benjamin, H. Potter, D. W. Yandell, and J. B. Little, Proc. Natl. Acad. Sci. USA 88:6652-6656, 1991). This cell line (designated 6:86) carries unique heterozygous frameshift mutations in exons 4 and 7 of its endogenous tk alleles and can revert to TK+ by frame-restoring mutations, gene conversion, or reciprocal recombination. Line 6:86 reverts spontaneously to TK+ at a frequency of 10-7 to 10-8, and exposures to X-irradiation or the frameshift mutagen ICR-191 induce increased reversion frequencies in a dose-dependent manner. Another cell line (designated 4:2) carries a homozygous exon 7 frameshift and is not expected to revert through mechanisms other than frame-restoring mutation. Line 4:2 reverts to TK+ at a lower spontaneous frequency than does 6:86 but can be induced with similar kinetics by ICR-191. In contrast to line 6:86, however, X rays did not induce detectable reversion of line 4:2. We have characterized a number of 6:86-derived revertants by means of restriction fragment length polymorphism analysis at tk and linked loci, single-strand conformation polymorphisms, and direct transcript sequencing. For X rays, most revertants retain both original mutations in the genomic DNA, and a subset of these frameshift-retaining revertants produce frameshift-free message, indicating that reversion is the result of reciprocal recombination within the tk gene. Frame-restoring point mutations, restoration of original sequences, and phenocopy reversion by acquisition of aminopterin resistance were also found among X-ray-induced revertants, whereas the ICR-191-induced revertants examined show only loss of the exon 7 frameshift.Molecular genetic analysis has yielded evidence to support a role for somatic interchromosomal homologous recombination in the genesis and progression of a number of human tumors (8,16,33,42,54). Evidence has been presented recently for spontaneous somatic recombination in cells from patients with cancer-predisposing Bloom's syndrome (24,36), and this process has also been implicated in the apparent polyclonal origin of some human tumors (65). The very low frequency with which human somatic chromosomes appear to undergo homologous recombination has made the study of this phenomenon particularly difficult. The use of plasmid-based extrachromosomal recombination assays (for a review, see reference 11; 39, 50, 60, 71, 72), recombination between tandemly arranged genes integrated into mammalian chromosomes (for reviews, see references 11 and 34; 3, 9, 10, 28-30, 69), or recombination between transfected and endogenous sequences (for a review, see reference 11; 1, 5, 47, 64) has proven to be effective for analyzing both the mechanisms and induction of mammalian somatic recombination. (RFLPs) are then used to distinguish hemizygotes from homozygotes and nondisjunctional loss from interchromosomal recombination. Only one product...

Epidemiologic application of the human in vivo hypoxanthine-guanine phosphoribosyltransferase (hprt) mutation assay requires screening of mutant colonies to differentiate independent from clonal origin. Previously, sibship was defined by Southern blot analysis of T cell receptor gene rearrangements. We report here a more expedient method to determine these rearrangements utilizing the polymerase chain reaction (PCR) and a DNA single-strand conformation polymorphism technique. The results are consistent with those obtained by Southern blotting in that sibship can be defined easily. A major advantage is that cells may be taken directly from the microtiter plate, eliminating the necessity to expand the clones and isolate genomic DNA. Cell lines which have not undergone receptor gene rearrangements cannot serve as PCR templates and do not interfere with this analysis. Furthermore, background from the large number of nonmutant lymphocytes present in the well does not hinder the analysis of the T cell receptor pattern of a mutant. This technique facilitates rapid screening of a large number of clones in a shorter time than Southern blotting, and is useful for the study of in vivo mutation and the clonal expansion of mutants in populations of T cells.

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We have developed a human lymphoblast cell line for the study of interchromosomal homologous recombination at the endogenous thymidine kinase (tk) gene on chromosome 17 (M. B. Benjamin, H. Potter, D. W. Yandell, and J. B. Little, Proc. Natl. Acad. Sci. USA 88:6652-6656, 1991). This cell line (designated 6:86) carries unique heterozygous frameshift mutations in exons 4 and 7 of its endogenous tk alleles and can revert to TK+ by frame-restoring mutations, gene conversion, or reciprocal recombination. Line 6:86 reverts spontaneously to TK+ at a frequency of 10(-7) to 10(-8), and exposures to X-irradiation or the frameshift mutagen ICR-191 induce increased reversion frequencies in a dose-dependent manner. Another cell line (designated 4:2) carries a homozygous exon 7 frameshift and is not expected to revert through mechanisms other than frame-restoring mutation. Line 4:2 reverts to TK+ at a lower spontaneous frequency than does 6:86 but can be induced with similar kinetics by ICR-191. In contrast to line 6:86, however, X rays did not induce detectable reversion of line 4:2. We have characterized a number of 6:86-derived revertants by means of restriction fragment length polymorphism analysis at tk and linked loci, single-strand conformation polymorphisms, and direct transcript sequencing. For X rays, most revertants retain both original mutations in the genomic DNA, and a subset of these frameshift-retaining revertants produce frameshift-free message, indicating that reversion is the result of reciprocal recombination within the tk gene. Frame-restoring point mutations, restoration of original sequences, and phenocopy reversion by acquisition of aminopterin resistance were also found among X-ray-induced revertants, whereas the ICR-191-induced revertants examined show only loss of the exon 7 frameshift.