Worm Breeder's Gazette 12(3): 92 (June 15, 1992)

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.

Forced Expression of the Tc3 ORF ( Tc3A )Results in Tc3 Excision

Henri G.A.M. van Luenen, Ronald H.A. Plasterk

The Netherlands Cancer Institute, Division of Molecular Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

We study the mechanism of Tc3 transposition. To test the role of the putative transposase Tc3A ,the ORF encoded by the Tc3 element isolated by Collins et al. (Genetics 121:47-55 (1989)), we expressed the protein in the worm.

Constitutive expression of a transposase might be lethal for the organism. Therefore we used an inducible promoter system: a heat shock promoter ( hsp-16 )(Russnak, R.H. and E.P.M. Candido, Mol. Cell. Biol. 5:1268-1278 (1985)). The intergenic region between hsp 16-1 and hsp 16-48 from PC31 (kindly provided by Dr. Candido) has been fused to the ORF of Tc3 .This construct, together with rol-6 has been injected in Bristol N2 .Expression of the hsp- Tc3A construct is induced by placing the plates for 2 hours at 33°C.

RNA has been isolated from 4 independent transgenic lines after a heat shock. In all cases a strong Tc3 specific signal could be detected on a Northern blot. Without heat shock we do not see a Tc3 specific transcript. From the same heat shocked worm cultures total protein extracts were made. Using antisera directed against the 13 most C- or N-terminal amino acids we were able to detect the Tc3A protein after induction.

Next we determined whether expression of Tc3A in vivo leads to Tc3 activity. First we looked at general survival of transgenic animals after a heat shock. If Tc3A expression would lead to excision and transposition this might be lethal for the animal, since very frequent somatic transposition may result in chromosomal damage. 10 L2 larvae from either Bristol N2 or two independent transgenic lines have been heat shocked. All the N2 larvae complete development and produce offspring. 9 out of 10 larvae from one transgenic line and 8/10 larvae from the other line did not complete development and produced no offspring. When the strains were not submitted to a heat shock all larvae developed and produced offspring. Adult animals from both N2 and the two transgenic lines survived the treatment

We also tried to detect somatic excision of Tc3 after induction of Tc3A .Undigested genomic DNA from induced and non-induced cultures were run on an agarose gel and blotted onto nitrocellulose. When probed with a Tc3 fragment, extrachromosomal Tc3 copies could be detected 30 minutes after the heat shock. The increase levels off after approximately 1 hour of recovery at room temperature. Several minor bands can been seen which probably represent different topological forms of circular extrachromosomal Tc3 elements. The extrachromosomal Tc3 copies will be examined further. We determined that expression of Tc3A in C. elegans leads to Tc3 excision. Now that we know that Tc3A encodes a functional protein we will purify the protein and study the transposition reaction in vitro.

Literature Cited:

Collins et al. Genetics 121:47-55 (1989)

Russnak, R.H. and E.P.M. Candido, Mol. Cell. Biol. 5:1268-1278 (1985)