DETECTION OF CIRCULAR EXCISED DNA DELETION ELEMENTS IN TETRAHYMENA-THERMOPHILA DURING DEVELOPMENT

Extensive programmed DNA deletion occurs in ciliates during developmen t. In this study we examine the excised forms of two previously charac terized deletion elements, the R- and M-element, in Tetrahymena. Using divergently oriented primers in polymerase chain reactions we have de tected the junctions formed by joining the two ends of these elements, providing evidence for the presence of circular excised forms. These circlar forms were detected in developing macronuclear DNA from 12-24 h after mating began, but not in micronuclear or whole cell DNA of veg etative cells. They are present at very low abundance, detectable afte r PCR only through hybridization with specific probes. Sequence analys is shows that the circle junctions occur at or very near the known end s of the elements. There is sequence microheterogeneity in these junct ions, which does not support a simple reciprocal exchange model for DN A deletion. A model involving staggered cuts and variable mismatch rep air is proposed to explain these results. This model also explains the sequence microheterogeneity previously detected among the junction se quences retained in the macronuclear chromosome.