Mapping the polarity of changes that occur in interrupted CAG repeat tracts in yeast.

To explore the mechanisms by which CAG trinucleotide repeat tracts undergo length changes in yeast cells, we examined the polarity of alterations with respect to an interrupting CAT trinucleotide near the center of the tract. In wild-type cells, in which most tract changes are large contractions, the changes that retain the interruption are biased toward the 3' end of the repeat tract (in reference to the direction of lagging-strand synthesis). In rth1/rad27 mutant cells that are defective in Okazaki fragment maturation, the tract expansions are biased to the 5' end of the repeat tract, while the tract contractions that do not remove the interruption occur randomly on either side of the interruption. In msh2 mutant cells that are defective in the mismatch repair machinery, neither the small changes of one or two repeat units nor the larger contractions attributable to this mutation are biased to either side of the interruption. The results of this study are discussed in terms of the molecular paths leading to expansions and contractions of repeat tracts.

Orientation dependence of trinucleotide CAG repeat instability in Saccharomyces cerevisiae.

To examine the chromosomal stability of repetitions of the trinucleotide CAG, we have cloned CAG repeat tracts onto the 3' end of the Saccharomyces cerevisiae ADE2 gene and placed the appended gene into the ARO2 locus of chromosome VII. Examination of chromosomal DNA from sibling colonies arising from clonal expansion of strains harboring repeat tracts showed that repeat tracts often change in length. Most changes in tract length are decreases, but rare increases also occur. Longer tracts are more unstable than smaller tracts. The most unstable tracts, of 80 to 90 repeats, undergo changes at rates as high as 3 x 10(-2) changes per cell per generation. To examine whether repeat orientation or adjacent sequences alter repeat stability, we constructed strains with repeat tracts in both orientations, either with or without sequences 5' to ADE2 harboring an autonomously replicating sequence (ARS; replication origin). When CAG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively stable regardless of the orientation of ADE2. When CTG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively unstable regardless of the orientation of ADE2. Removal of the ARS as well as other sequences adjacent to the 5' end of ADE2 alters the orientation dependence such that stability now depends on the orientation of ADE2 in the chromosome. These results suggest that the proximity of an ARS or another sequence has a profound effect on repeat stability.

Infection after intramedullary nailing of severe open tibial fractures initially treated with external fixation.

Twenty-four patients had a severe open fracture of the tibia that was initially treated by external fixation and subsequently by reamed intramedullary nailing. The external fixation had been maintained for an average of fifty-two days (range, seven to 230 days). The mean interval between removal of the external fixator and intramedullary nailing was sixty-five days (range, three to 360 days). In five of the seven patients who had had an infection at one or more of the pin sites, an infection later developed around the intramedullary nail. In comparison, only one of the seventeen patients who had not had a pin-site infection had an infection later around the nail (p = 0.003). An analysis of other variables, including the duration of external fixation, wound coverage, other injuries, and the type of fracture, showed that none was a predictor of infection either at the pin sites or around the intramedullary nail. We concluded that a pin-site infection that develops during external fixation is a contraindication to the subsequent use of reamed intramedullary nailing in patients who have a fracture of the tibia.