Direct cloning and analysis of DNA sequences from a region of the Chinese hamster genome associated with aphidicolin-sensitive fragility

Abstract

Fragile sites are reproducibly expressed and chemically induced decondensations on mitotic chromosomes observed under cytological conditions. They are classified both on the basis of the frequency with which they occur (rare and common) and in terms of the chemical agent used to induce expression in tissue culture cells. Aphidicolin-sensitive common fragile sites appear to be ubiquitous in humans and other mammals and have been considered as candidates of pathological importance. Recently DNA from FRA3B, the most highly expressed constitutive fragile site in the human genome, has been cloned although as yet the cause of the underlying fragility has not been identified. In this study we describe the isolation, using a direct cloning approach, of DNA from a region of the Chinese hamster genome associated with aphidicolin-inducible fragility. Cells of a human-hamster somatic cell hybrid were transfected with a pSV2HPRT vector while exposed to aphidicolin, an inhibitor of DNA polymerases alpha, delta and epsilon. FISH analysis of stable transfectant clones revealed that the ingoing plasmid DNA had preferentially integrated into fragile site-containing chromosomal bands. Plasmid rescue was used to recover DNA sequences flanking one such integration site in the hamster genome. We demonstrate by FISH analysis of metaphase cells induced with aphidicolin that the rescued DNA is from a region of fragility on Chinese hamster chromosome 2, distal to the DHFR locus. Analysis of the DNA sequences flanking the integration site revealed the overall A+T content of the 3,725 bp region sequenced to be 63.3%, with a highly [A].[T]-rich 156 bp region (86.5%) almost adjacent to the integration site. Computational analyses have identified strong homologies to Saccharomyces cerevisiae autonomous replicating sequences (ARS), polypyrimidine tracts, scaffold attachment site consensus sequences and a 24 bp consensus sequence highly conserved in eukaryotic replication origins, all of which appear to cluster around the [A].[T]-rich sequences. This domain also possesses structural characteristics which are common to both prokaryotic and eukaryotic origins of replications, in particular an unusually straight conformation of low thermal stability flanked either side by highly bent DNA segments. Further isolation and characterisation of DNA sequences from common fragile sites will facilitate studies into the underlying nature of these enigmatic regions of the mammalian genome, leading to a greater understanding of chromatin structure.