Recombination and deletion of sequences in shuttle vector plasmids in mammalian cells.

Abstract

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.

Efficient homologous recombination of linear DNA substrates after injection into Xenopus laevis oocytes.

“…In all systems studied to date, double-strand breaks in one or both participating substrates have been found to stimulate the frequency of recombination (6,9,22,24,30,34,39,49,56). This may be a reflection of a natural role for recombination in the repair of damage to DNA.…”

Intermolecular recombination assay for mammalian cells that produces recombinants carrying both homologous and nonhomologous junctions.

“…The main conclusions from these assays are that cultured cells will readily recombine exogenous DNA by homologous (6,11,17,20,27,(33)(34)(35) and nonhomologous recombination (10,12) and that when compared in intramolecular assays, both recombination mechanisms have similar frequencies (6,31,37,39). The frequency of homologous recombination depends on the length of homology (3,18,21,32,34,39), and it can be significantly increased by introducing a doublestrand break in or near the selected homologous sequences (5,6,11,20,21,33,49).…”

“…The frequency of homologous recombination depends on the length of homology (3,18,21,32,34,39), and it can be significantly increased by introducing a doublestrand break in or near the selected homologous sequences (5,6,11,20,21,33,49). This has prompted several authors to propose models for homologous recombination involving a double-strand break (21,44,49).…”

“…This has prompted several authors to propose models for homologous recombination involving a double-strand break (21,44,49). Double-strand breaks have also been reported to increase illegitimate recombination (6,12), which is thought to involve ligation of free ends (12,48).…”

“…In most cases, the recombination products are identified by the reconstitution of a selectable marker, as a result of either intra-or intermolecular homologous recombination (2, 3, 5, 8, 11, 12, 17, 20-24, 27, 28, 32-36, 38, 40, 45, 47, 49, 50). Some assays have also looked at nonhomologous recombination (1,13,19,30,52), and in a few instances, intramolecular recombination assays have been devised that permit comparison of homologous and nonhomologous recombination (6,31,37,39).…”