Several possible strategies for the cloning of new FA genes exist. cDNA complementation of the DNA cross-linking hypersensitivity has already resulted in the cloning of one FA gene. This method, however, is limited by 2 factors: a) the cDNA has to be relatively small and b) properly regulated expression of the gene must not he essential for cell survival. An alternative method for isolating FA genes is positional cloning. Due to the fact, that multiple complementation groups exist and that the number of families is relatively limited, the mapping of the FA genes by genetic linkage studies alone appears difficult We here propose to use chromosome transfer to map the location of FA genes at a gross level, followed by genetic linkage and physical mapping to further refine the chromosomal position and to ultimately clone the genes. Human chromosomes marked with the 0418 resistance gene will be transferred into transformed fibroblasts from FA patients, which are known to not represent complementation group C. Selection with DNA crosslinking agents will identify complemented clones and the chromosome conferring the resistance will be identified. The size of the correcting cDNA is not relevant in this method and proper regulation of the gene in question is also given. Once a complementing chromosome has been identified, the more precise localization of the FA gene will be identified by deletion mapping in somatic cell hybrids and genetic linkage. Existing markers as well as new markers generated by chromosome microdissection and PCR will be employed for genetic mapping. This step will be followed by isolation of YACs from the critical region, physical mapping and the cloning of expressed sequences. Candidate cDNAs will be screened by chemical mismatch cleavage for possible mutations. An additional approach to be used is the identification of mouse chromosomes bearing FA genes as described above. FA cells complemented by mouse DNA could then be used to prepare subtractive cDNA libraries and direct cloning of FA candidate cDNAs.

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