GENOMIC PLASTICITY IN THE HUMAN U2 SNRNA GENE CLUSTER

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Gene amplification and chromosome fragility play important roles in human genetics but the molecular mechanisms responsible for such genomic plasticity are not well understood. Two remarkable features of the multigene family encoding human U2 small nuclear RNA (snRNA) make it ideal for studying both amplification and fragility at the molecular level. First, in contrast to most multigene families, the 10 to 20 human U2 genes are organized in an apparently perfect tandem array; each copy of the 6 kb repeat unit is identical in sequence, and RFLPs are rare or nonexistent. This implies that the homogeneity of the repeats is maintained by a rectification process, or that the cluster itself is young and was originally generated by a mechanism capable of producing perfect tandem repeats from only one or a few initial gene copies. In either case, the repeat unit itself and/or the sequences flanking the tandem array are likely to contain recombinogenic elements that promote rectification, generation, or regeneration of the cluster. Our first major goal is to identify such recombinogenic sequences. The second remarkable feature of the U2 tandem array is that the cluster maps to a site of chromosomal fragility induced by oncogenic strains of adenovirus. In fact, Durnam et al. (1988) have recently shown that the fragile site is highly localized and that breakage often occurs within the U2 gene cluster itself (i.e. within a region of only 60 to 120 kb). Thus the U2 cluster is likely to contain elements that cause chromosomal fragility during adenovirus infection. Our second major goal is to identify these fragility inducing elements. We intend to identify both the recombinogenic elements and the fragility inducing elements by screening the U2 array for sequences which promote gene amplification and/or chromosome fragility when integrated into new chromosomal sites. Cell lines containing all or part of the U2 6kb repeat unit adjacent to a dihydrofolate reductase (DHFR) minigene will be constructed by cotransfection. Recombinogenic elements will be identified by scoring the frequency of spontaneous or induced DHFR amplification by cell sorting or by methotrexate selection. In cell lines having chromosomally amplified DHFR genes, the ability of coamplified U2 sequence elements to confer fragility on the integration site will be assayed after superinfection with adenovirus or transfection with the El region of the virus.