UNEXPECTEDLY LOW-LOSS OF HETEROZYGOSITY IN GENETICALLY UNSTABLE WERNER SYNDROME CELL-LINES

We have determined the mitotic stability of micro- and mini-satellite DNA sequences in SV40-immortalized Werner syndrome (WS) and control fi broblast cell lines. Five microsatellite loci were genotyped in two WS and two control SV40-immortalized fibroblast cell lines and in 154 in dependent primary or secondary clones derived from these. We used four minisatellite ''core'' or individual locus probes in Southern blot hy bridization analyses to assess minisatellite stability in WS and contr ol clones. Microsatellite allele length was stably maintained in both WS and control cells, and an upper limit for the generation of new all ele lengths was estimated to be less than or equal to 4.5 x 10(-4)/all ele/generation (or less than or equal to 2.25 x 10(-5)/CA repeat/gener ation). In contrast to length stability, loss of heterozygosity (LOH) at microsatellite loci ranged up to 76% at the 13 informative locus:ce ll line combinations. An unexpected, and counterintuitive, finding was a much lower frequency of LOH in WS than in control clones at microsa tellite loci on three different chromosomes. Minisatellite band altera tions (gains, losses, or band intensity differences) were 4-fold lower in WS than in control cells. Our results suggest that the chromosomal and molecular genetic instability displayed by WS cells is unlikely t o be the result of a micro- or mini-satellite destabilizing defect. A second, unexpected conclusion is that WS cells may possess a novel mea ns of either suppressing or masking LOH events in the presence of cons titutional cytogenetic and molecular genetic instability. (C) 1997 Wil ey-Liss, Inc.