Mitotic recombination produces the majority of recessive fibroblast variants in heterozygous mice - PubMed (original) (raw)

Mitotic recombination produces the majority of recessive fibroblast variants in heterozygous mice

C Shao et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Mice heterozygous at Aprt (adenine phosphoribosyltransferase) were used as a model to study in vivo loss of heterozygosity (LOH) in normal fibroblasts. Somatic cell variants that exhibited functional loss of the wild-type Aprt in vivo were recovered as APRT-deficient cell colonies after culturing in selection medium containing 2, 6-diaminopurine (DAP), an adenine analog that is toxic only to cells with APRT enzyme activity. DAP-resistant (DAP(r)) fibroblast variants were recovered at a median frequency of 12 x 10(-5) from individual ears from progeny of crosses between mouse strains 129/Sv and C3H/HeJ. The frequency of DAP(r) variants varied greatly among individual ears, suggesting that they preexisted in vivo and arose at various times during development. Polymorphic molecular markers and a cytological marker on the centromere of chromosome 8 made it possible to discriminate between each of six possible mechanistic pathways of LOH. The majority (about 80%) of the DAP(r) variants were a consequence of mitotic recombination. The prevalence of mitotic recombination in regions proximal to Aprt did not correlate with meiotic map distances. In particular, there was a higher than expected frequency of crossovers within the interval 59 cM to 67 cM. The high spontaneous frequency of Aprt LOH, mediated primarily by mitotic recombination, is fully consistent with our previous results with human peripheral T cells from individuals known to be heterozygous at APRT. Thus, this Aprt heterozygote mouse is a valid model for studying somatic mutagenesis and mitotic recombination in vivo.

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Figures

Figure 1

Figure 1

Allele-specific PCR of representative DAPr clones. Clones 2, 3, 4, 6, 7, 9, 10, 11, and 12, designated as class I, exhibited physical loss of Aprt+. Clones 1, 5, and 8, designated as class II, retained Aprt+.

Figure 2

Figure 2

Intervals of LOH in class I clones. The lines correspond to the interval for which the SSR markers remained heterozygous. All markers right to the lines exhibited LOH. The number of independent events in each group is shown on the left. The map positions of the SSR markers are according to Mouse Genome Database [

http://www.informatics.jax.org

(6/98)].

Figure 3

Figure 3

Cytogenetic evidence that LOH was caused by mitotic recombination. (A) Whole-chromosome painting of a class I DAPr fibroblast clone. Chromosome regions exhibiting hybridization signal are green, otherwise they stain red. Chromosome 8 of strain C3H/HeJ (Chr8C3H) exhibits a large centromeric region. Although SSR genotyping showed a large interval of LOH (loss of C3H alleles), the homologue with a large centromere, Chr8C3H, exhibited no corresponding terminal deletion. (B) Aprt FISH of a class I DAPr clone. SSR genotyping showed terminal LOH (loss of C3H alleles) beginning at 59 cM, but Aprt hybridization signals were evident on both Chr8129 and Chr8C3H.

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