Circular forms of developmentally excised DNA in Euplotes crassus have a heteroduplex junction

Extensive DNA elimination via a DNA breakage and rejoining process occurs during macronuclear development in the hypotrich ciliate Euplotes crassus. The excision process involves the removal of short, unique segments of DNA (internal eliminated sequences; IESs) and at least two highly repetitive families of transposon-like elements (Tec elements). Previous studies have demonstrated that circular forms of both IESs and Tec elements are generated following their developmental excision and that some flanking DNA sequences are retained at the circle junctions. In this study we have further analyzed the circle junctions of IESs. Analysis of polymerase chain reaction (PCR) products derived from IES circle junctions indicates that at least two sequence arrangements can be present. The circle junctions contain both of the direct repeats that define the ends of the IES separated by either 2 bp flanking the right end of the IES and 8 bp from the left-flanking region, or 8 bp from the right and 2 bp from the left. Using a method that we have termed "strand-biased PCR," we obtained evidence that the junctions of free circular IESs have a 6-base heteroduplex at their center, such that one strand of the DNA is derived from the left-flanking region of the IES and the other from the right. Models of IES excision are presented that incorporate these results and those of previous studies on the excision process.