Worm Breeder's Gazette 11(5): 46
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.
Recent studies of transposon Tc1 have focused on the Tc1 open reading frame product, TcA, and its function in transposition. Since different levels of transposition activity have been detected in different worm strains and tissues, the cellular environment appears to influence the transposon function. Unpublished data from our laboratory have shown that Tc1 transcripts are about 1.3 Kb in length and start 5' to the ORF at multiple sites between 137 and 380 bp ( Radice & Emmons, per. comm.). We propose that transposition activity may correlate with Tc1 transcription activity. In this study, we examined the 5' internal sequence and flanking region of Tc1 tor interaction with cellular factors in order to see whether transcriptional signals and activity can be identified in the transposon or its flanking sequence. We subcloned a 258 bp fragment containing 138 bp of the 5' end of Tc1 element plus 120 bp of flanking DNA from a genomic clone pCE2001 ( see figure), and examined the association of this fragment with cellular factors by gel retardation assays and DNase I . A worm whole cell extract was made from the C. elegans Bergerac strain, which is active tor both Tc1 somatic excision as well as germline transposition and excision. Using from 1 to 20 g of extract protein tor each assay, we were able to detect substantial binding activity to the Tc1 DNA probe in the extract. However, competition with specific and non- specific cold DNA showed that the binding activity was not specific to the Tc1 probe. DNase I footprinting gave a more promising result. Using probes of the same 258 bp fragment labelled at either end and a whole cell extract, we were able to identify a number of protected regions and DNase I hypersensitive sites (see figure). The protected regions were mapped to sequences outside Tc1 in the flanking DNA. Two DNase I hypersensitive sites were detected in the presence or absence of protein binding (+/-), one near the end of the Tc1 element and one inside the element. An additional hypersensitive site within the element (+) was detected only in the presence of protein extract. This additional hypersensitive site strongly argues for specific DNA binding activity in the extract which can cause DNA conformational changes to the Tc1 sequence. Currently, we are trying to further define the binding regions. We are also aware of the DNA binding activity of transposase TcA and have analyzed the specificity of its binding to the Tc1 sequence. We adopted an immunoprecipitation approach using mixed labelled DNA fragments containing plasmid sequences or Tc1 sequences. DNA was incubated with either worm whole cell extract, E. coli extract or extract from E. coli expressing TcA to allow interaction with DNA binding protein. Incubation was followed by addition of purified antibody against TcA (from Ron Plasterk), and then protein A sepharose which interacts with the Ab. A series of washes with buffer eliminated any nonspecific binding of DNA to the protein recognized by the Ab (presumably TcA). Running the remaining bound fragments on an agarose gel revealed the specificity of the binding activity. The result showed that the binding activity recognized by the Ab was present in the worm extract and in the extract of E. coli expressing TcA but absent in the extract of E. coli not expressing TcA. This result indicated that we were examining the binding activity of TcA in E. coli and suggested that the same was true for the worm extract. This would be the first demonstration of the presence of TcA in worms. The activity however was nonspecific. All the DNA fragments, with or without Tc1 sequences, were precipitated. It therefore supports the finding of Ron Plasterk that TcA is a non-specific DNA binding protein. However, this does not eliminate the possibility that TcA, in concert with additional factors, is involved in the regulation of Tc1 transcription. To further investigate the potential transcriptional activity associated with the 5' region of Tc1, we have made a reporter construct linking the -galactosidase gene with a 750 bp Tc1 5' flanking and internal sequence from pCE2001, and will test for transcriptional activity by transformation into the worms. The result will give us some idea whether transcriptional signals are present in this DNA fragment. If so, deletion analysis will allow localization of these signals to the Tc1 or to flanking genomic sequences, and shed light on the possible control of transposon activity. [See Figure 1]