CELL AND SITE SPECIFIC DNA REARRANGEMENTS

DNA rearrangements have been found in both prokaryotic and eukaryotic genomes, leading to the surprising conclusion that the genome is far more dynamic that previously believed. While studying the 27kD zein locus in maize, we discovered that it is susceptible to several different novel DNA rearrangements. In inbred maize strains the locus exists either as a single gene or as a tandem duplication of 12 kb that includes the 670 bp coding region. In all cases examined thus far, the single gene was derived irreversibly from the duplication by homologous intrachromosomal recombination. Most of the recombinations occur somatically, leading to a mosaic of cells, although some can be introduced into the gametes and be inherited. In either case, the pattern rearrangement appears to be cell and site specific. Therefore, the tandem duplication should contain sequences that are recognized and regulated by specific recombinases. We would like to study this process and its role using a combination of molecular techniques and genetic approaches. In situ hybridization techniques will be used to study these events on the cellular level. Matings and backcrosses between several of the inbreds should allow us to distinguish between the different cellular factors that play a role in recombination, and whether all 27kD alleles can be affected. We have already begun cloning and sequencing the recombined and unrecombined loci isolated from different inbred strains, and from endosperm of the inbred with high-frequency rearrangements. Recombination in different cell types will be studied using a viral vector system, introduced into cell cultures and plant tissues with a particle gun. The recombined vector will be rescued in bacterial cells by a novel selection system that has proven effective in a similar analysis of the V-(D)-J recombination system in mammalian lymphocytes.