Reciprocal homologous junctions generated in mouse cells.

We analysed pairs of reciprocal homologous junctions resulting from intermolecular conservative homologous recombination in mouse cells. The assay used did not rely on the reconstitution of a selectable gene. This permitted the introduction of multiple markers in the parental homologous sequences which in turn enabled us to compare the contribution of each parent to the reciprocal products of a given recombination event. In all recombinants analysed we found, when comparing the reciprocal junctions, a middle segment originating from only one parent. This segment of uniparental origin occurred randomly throughout the region of homology and could extend over a thousand base pairs. These results are consistent with a gap repair process like the one proposed for homologous recombination in yeast. However, introducing a double-strand break in the region of homology did not enhance but rather decreased the proportion of recombinants with reciprocal homologous junctions relative to other types of recombinants.

Characterization of nonconservative homologous junctions in mammalian cells.

Homologous recombination in mammalian cells between extrachromosomal molecules, as well as between episomes and chromosomes, can be mediated by a nonconservative mechanism. It has been proposed that the key steps in this process are the generation (by double-strand cleavage) of overlapping homologous ends, the creation of complementary single-strand ends (either by strand-specific exonuclease degradation or by unwinding of the DNA helix), and finally the creation of heteroduplex DNA by the annealing of the single-strand ends. We have analyzed in detail the structure of nonconservative homologous junctions and determined the contribution of each end to the formation of the junction. We have also analyzed multiple descendants from single recombination events. Two types of junctions were found. The majority (90%) of the junctions were characterized by a single crossover site. These crossover sites were distributed randomly throughout the junction. The remaining 10% of the junctions had mosaic patterns of parental markers. Furthermore, in 9 of 10 cases, multiple descendants from a single recombination event were identical. Thus, it appears that in most cases few parental markers were involved in junction formation. This finding suggests that nonconservative homologous junctions are mediated mainly by short heteroduplexes of a few hundred base pairs or less. These results are discussed in terms of the current models of nonconservative homologous recombination.

Individualized aminoglycoside dosage regimens in patients with cystic fibrosis.

Individualized dosage regimens have recently been recommended for patients treated with aminoglycoside antibiotics. We have developed a calculator-based program for our patients with cystic fibrosis and have studied 93 courses of intravenous aminoglycoside treatment, comparing predicted and measured values in 45 courses. Pharmacokinetic parameters differed notably among subjects: this was reflected by widely variable total daily aminoglycoside dosage requirements. The mean daily dosage requirements (+/- SD) for tobramycin (62 treatment courses) was 13.0 +/- 3.74 mg/kg, and for gentamicin (26 treatment courses) was 11.5 +/- 2.6 mg/kg. The accuracy of the program was evaluated by its ability to predict peak and trough values in individuals: 84 percent of measured peaks were within 2 micrograms/ml of the predicted level. Nephrotoxicity was observed in one patient, ototoxicity in three. This program provides a simple, safe, and effective method of tailoring an aminoglycoside regimen to the patient's needs.