Worm Breeder's Gazette 8(2): 51
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Extrachromosomal linear and circular copies of transposable element Tc1 are present in Bergerac at an average level of 0.1-1.0 copies per cell. They can be detected as discrete species of high electrophoretic mobility when Bergerac DNA is fractionated on an agarose gel, transferred to nitrocellulose, and hybribized to a Tc1- specific probe. We purified them on sucrose gradients and studied their structure by analyzing the sizes of the products produced by treatment with various restriction endonucleases known to cleave within Tc1. The predominant species in our preparations (perhaps 75% of the material) was a linear copy of the transposon with non-permuted ends that corresponded exactly or very closely to the ends of inserted Tc1 elements. The next most abundant species (perhaps 20%) was a relaxed circular copy of the transposon. There was a third species (about 5%) which we showed was a supercoiled copy of the element, by CsCl-ETBr density gradient experiments, but which was present in amounts too low to analyze in detail. The structure of the ends of the linear molecules and of the end-to- end joint in the circular molecules is of particular interest because of the ambiguity in defining the ends of Tc1 from the sequencing results of Rosenzweig, Liao and Hirsh. They found that at two Bergerac-specific Tc1 insertion sites the nucleotides present in Bergerac but absent from Bristol could be interpreted as comprising a 1610 nucleotide-long Tc1 element and a two-nucleotide target-site duplication, or a 1612 nucleotide-long Tc1 element with no target-site duplication. The ambiguity could not be resolved because the two nucleotides comprising the putative target-site duplication were the same at both sites. When the Tc1 sequence as defined by each of these interpretations (plus and minus the final nucleotides) is circularized, each produces a different restriction site at the novel end-to-end joint. If the final nucleotides are not included (1610 nucleotide Tc1 element), a PstI site (CTGCAG) is created, while if the final nucleotides are included (1612 nucleotide Tc1 element), an RsaI site ( GTAC) is created. We found that the end-to-end joint in the circular molecules was cleaved by RsaI but not PstI. If extrachromosomal Tc1 elements are indeed products of excision, this result suggests that Tc1 consists of a 1612 nucleotide sequence and produces no target-site duplication upon integration. If Tc1 does produce a target-site duplication, then the additional assumptions must be made that most or all Tc1 elements are inserted at a TA dinucleotide and one T and one A are excised with the element in order to explain the results.