| dc.description.abstract | Previous studies in the laboratory have shown that a duplication of the C{dollar}\sb{lcub}\gamma\sp{lcub}\rm 2a{rcub}{rcub}{dollar} immunoglobulin heavy chain constant region gene has occurred on the expressed chromosome in the mouse myeloma cell line MPC-11. This duplication, along with the deletions and increases in copy number of the C{dollar}\sb{lcub}\gamma\sp{lcub}\rm 2a{rcub}{rcub}{dollar} gene observed in variants of MPC-11, arose via an unequal sister chromatid exchange (USCE). In order to gain further insight into the mechanisms involved in the USCE process, the regions {dollar}\sim{dollar}2.5 kb 3{dollar}\sp\prime{dollar} of the C{dollar}\sb{lcub}\gamma\sp{lcub}\rm 2b{rcub}{rcub}{dollar} gene ({dollar}\sb\gamma{dollar}2b-3{dollar}\sp\prime{dollar}) and {dollar}\sim{dollar}2.5 kb of the C{dollar}\sb{lcub}\gamma\sp{lcub}\rm 2a{rcub}{rcub}{dollar} gene ({dollar}\sb\gamma{dollar}2a-3{dollar}\sp\prime{dollar}) that were involved in the recombination event were subcloned and sequenced. It was found that both regions possessed a high degree of sequence identity. Moreover, both sequences possessed a homopurine-homopyrimidine (TC {dollar}\cdot{dollar} AG){dollar}\sb{lcub}\rm n{rcub}{dollar} tract juxtaposed to an alternating purine-pyrimidine (TG {dollar}\cdot{dollar} AC){dollar}\sb{lcub}\rm n{rcub}{dollar} tract. Because of the ability of such simple sequence tracts to adopt alternative non-B-DNA conformations, and the possibility that such alternative DNA conformations may be important as recombination intermediates, these sequences along with sequence of the USCE-joint recombination product were analyzed in vitro for possible alternative DNA conformations using a variety of approaches. These included probing with {dollar}\Lambda{dollar}-Co(DiP){dollar}\sb3\sp{lcub}3+{rcub}{dollar}, S1 nuclease digestion, two-dimensional gel electrophoresis, and chemical probing with osmium tetroxide and diethylpyrocarbonate. In summary, it was found that the (TG {dollar}\cdot{dollar} AC){dollar}\sb{lcub}\rm n{rcub}{dollar} tracts were capable of adopting the Z conformation when present in a supercoiled plasmid at pH 7.6. When supercoiled plasmids with these sequences were subjected to pH 5.0, it was found that the (TC {dollar}\cdot{dollar} AG){dollar}\sb{lcub}\rm n{rcub}{dollar} tracts were capable of adopting an intramolecular triplex structure. In addition, it appeared that the formation of either of these structures within a single plasmid molecule was mutually exclusive, with Z-DNA predominating at pH 7.6 and triplex predominating at pH 5.0; at an intermediate pH of 6.0, both structures appeared to be equally present throughout the plasmid population.;In order to explore the role of these sequences in homologous recombination, a recombination assay is being developed. A general recombination substrate has been designed that will enable a wide variety of sequences to be assayed for their recombination potential in murine cells. This assay and substrate should provide a means for testing the ability of these sequences to promote and/or direct homologous recombination events. | |
