DIRECT CLONING OF DNA-SEQUENCES FROM THE COMMON FRAGILE SITE REGION AT CHROMOSOME BAND 3P14.2

Despite several lines of evidence suggesting that common chromosomal f ragile sites are biologically important as hot spots for recombination , their structure remains unknown. We showed previously that the plasm id pSV2neo preferentially integrates into bands containing fragile sit es in cells transfected under conditions of fragile site induction. He re we report the isolation and characterization of the DNA sequences f rom two such independent integrations into 3p14.2, a common fragile si te (FRA3B). These FRA3B region sequences were shown to lie within a 13 30-kb YAC, 850A6, approximately 350 kb telomeric of the breakpoint of t(3;8), a constitutional rearrangement. The two integration sites are 10 kb apart, but each integration is associated with a deletion. We ha ve constructed a partial genomic contig of the integration sites and d eleted regions spanning approximately 85 kb, Analysis of the DNA seque nces immediately surrounding the plasmid integrations revealed no know n coding sequences or repeat structures resembling the (CGG)(n) motif characteristic of the rare fragile sites. In addition, by Southern blo tting analysis, none of the phage clones isolated from the FRA3B regio n were found to contain CGG repeats. Fluorescence in situ hybridizatio n analysis of genomic clones from this contig to metaphase cells induc ed to express breaks demonstrated hybridization adjoining the chromoso me breaks, and occasionally the hybridization signal spanned the break . The results imply that breakage occurs at variable positions within a large region (at least on the order of 85 kb). Together, these data suggest that the structure of FRA3B differs from that of rare fragile sites. (C) 1996 Academic Press, Inc.