When plasmids carrying a fragmented gene with segments present as direct repeats are introduced into mammalian cells, recombination or gene conversion between the repeated sequences can reconstruct the gene. Intramolecular recombination leads to the deletion of the intervening sequences and the loss of one copy of the repeat. This process is known to be stimulated by double-strand breaks. Two current models for recombination in eucaryotic cells propose that the reaction is initiated by double-strand breaks, but differ in their predictions as to the fate of the intervening sequences. One model suggests that these sequences are always lost, while the other indicates that the reaction will be conservative as a function of the position of the double-strand break. We have constructed a plasmid in which two overlapping portions of the simian virus 40 early region, which contains the origin and T-antigen gene, are present as direct repeats separated by sequences containing a plasmid with a simian virus 40 origin of replication. Recombination across the repeated segments could produce a plasmid with an origin of replication and/or a plasmid with a gene for a functional T-antigen which would drive the replication of both. Introduction of this construction into African green monkey kidney cells, without coinfection, establishes a condition in which the products of the recombination or gene conversion can be interpreted unambiguously. We find that the majority of the reconstruction reactions are nonconservative.Recombination and gene conversion in mammalian cells have been studied by many groups who have monitored the reconstruction of selectable genes after infection with appropriately constructed viral or plasmid substrates (1, 3, 5-7, 11, 12, 17, 20-22, 24, 25, 27). The results of these experiments indicate that cells efficiently support both intra-and intermolecular recombination and gene conversion. Intramolecular recombination, the particular concern of this report, can be modeled with plasmid constructions in which duplicated regions of a fragmented (or otherwise defective) gene are present as direct repeats separated by some intervening sequences. Recombination between the repeated sequences produces a functional gene while deleting the intervening sequences (4,18). In experiments of this type, the focus is on the appearance of the active gene, and generally the fate of the intervening sequences is undetermined. Although there is limited experimental evidence on this point, the molecular models of recombination proposed by several groups do make specific predictions as to the fate of these sequences. Those models which suggest that recombination is initiated by nicks in a strand in one (15) or both (10) regions of homology predict that crossover resolution of the resultant Holliday junctions yields two smaller plasmids, each with one copy of the repeated sequence. Thus, the reaction, whatever the precise mechanism, would be conservative, i.e., the intervening sequence and both copies of the repeated element surviv...
Shuttle vector plasmids were constructed with directly repeated sequences flanking a marker gene. African green monkey kidney (AGMK) cells were infected with the constructions, and after a period of replication, the progeny plasmids were recovered and introduced into bacteria. Those colonies with plasmids that had lost the marker gene were identified, and the individual plasmids were purified and characterized by restriction enzyme digestion. Recombination between the repeated elements generated a plasmid with a precise deletion and a characteristic restriction pattern, which distinguished the recombined molecules from those with other defects in the marker gene. Recombination among the following different sequences was measured in this assay: (i) the simian virus 40 origin and enhancer region, (ii) the AGMK Alu sequence, and (iii) a sequence from plasmid pBR322. Similar frequencies of recombination among these sequences were found. Recombination occurred more frequently in Cosl cells than in CV1 cells. In these experiments, the plasmid population with defective marker genes consisted of the recombined molecules and of the spontaneous deletion-insertion mutants described earlier. The frequency of the latter class was unaffected by the presence of the option for recombination represented by the direct repeats. Both recombination and deletion-insertion mutagenesis were stimulated by double-strand cleavage between the repeated sequences and adjacent to the marker, and the frequency of the deletion-insertion mutants in this experiment was again independent of the presence of the direct repeats. We concluded that although recombination and deletion-insertion mutagenesis were both stimulated by double-strand cleavage, the molecules which underwent the two types of change were drawn from separate pools.
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