Recurring chromosomal abnormalities are characteristic of human malignant diseases. It is often assumed that chromosomal breaks occur randomly and that the chromosomal abnormalities noted in cancer cells result from selection of those rare changes that provide the cell with a selective advantage. An alternative possibility is that the frequency of specific chromosomal abnormalities may be influenced by variability in the propensity of various chromosomal sequences to undergo recombination. Under various culture conditions, chromosomal breaks and gaps occur at high frequency at specific chromosomal bands called fragile sites. Chromosomal fragile sites are highly recombinogenic, and fragile site expression leads to structural rearrangements. These observations suggest a role for fragile sites in the genesis of chromosomal abnormalities associated with cancer. The objective of this proposal is to characterize the common fragile site at 3p14, and to determine the mechanism(s) responsible for its propensity to undergo recombination. We have developed an experimental system in which exogenous DNA sequences preferentially integrate into chromosomal fragile sites following the induction of these sites. Moreover, integration of vector sequences at the fragile site results in an increase in genetic instability, and structural chromosome abnormalities involving this chromosome band. Recently we have used our experimental system to isolate the DNA sequences located at this fragile site. Using the cloned sequences, we will determine the size of the recombinogenic region as well as whether there are genes within the fragile site region. We will also determine the timing of replication of the fragile site, and whether its replication is delayed by agents which induce fragile sites. We will reintroduce the cloned DNA sequences into mammalian cells to determine whether chromosomal fragility is established at their sites of integration and, if so, we will identify the regions of the cloned sequences that are responsible for conferring this fragility. We hope also to begin to examine the mechanism of the increased fragility resulting from integration of exogenous DNA sequences. At present, the biological basis of the fragile site phenomenon is poorly understood. We hope that by addressing many of the biological questions related to fragile sites, we can evaluate their role in carcinogenesis in future studies.
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