Worm Breeder's Gazette 12(4): 15 (October 1, 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 Somatic Tc3 Transposition

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 (Collins et al. Genetics 121:47-55 (1989)),we expressed the protein in the worm using the heat shock inducible promoter (Stringham et. al. Mol. Biol. Cell 3:21-33 (1992)).

In the previous WBG (WBG 12/3:92 (1992)) we described the transgenic animals and the effect of forced expression of Tc3A on survival of L2 larvae. After the induction of Tc3A extrachromosomal Tc3 could be detected, indicating that Tc3A production results in excision of Tc3 .

(1) We analyzed Tc3 excision in more detail. After a two hour heat shock and approximately one hour of recovery at room temperature the highest level of extrachromosomal Tc3 is reached. At this point we estimate that there are approximately 0.6 extrachromosomal Tc3 elements per cell. At least 90 % of these elements have a linear structure, and are colinear with the integrated copy (determined by restriction mapping). This shows that Tc3A expression results in Tc3 excision by two double strand DNA breaks at the ends of integrated Tc3 copies.

(2) We extended the analysis of the transgenic animals in order to determine whether forced expression of Tc3A also results in Tc3 insertions. Therefore we determined the frequency of somatic Tc3 insertions in the gpa-2 gene. Somatic Tc3 insertions can be detected after PCR. In the non-transgenic strain Bristol N2 we found no Tc3 insertions in gpa-2 after a two hour heat shock and a subsequent two hour recovery period in 18 independent DNA samples (200 ng of total DNA each). In 18 DNA samples from a transgenic line which had continuously been grown at 18°C (in other words: not induced to produce Tc3A )we found 4 gpa-2 insertions. This is due to the constitutive, very low level of Tc3A production using this promoter. After a two hour heat shock 4 insertions where found and after a subsequent two hour recovery period we found 13 insertions. In the non-transgenic segregants of this line we found no insertions after a heat shock and recovery period. Most of the insertions have been sequenced and all were found to be genuine Tc3 insertions based on the criteria that the complete Tc3 ends were present and that the insertions take place at TA sequences. We conclude from these results that Tc3A production results in somatic integrations.

(3) Thus far we have not observed germ line integration of Tc3 in the progeny of heat shocked animals. This could be explained by the lack of expression of this transgene in the germ line.

Overexpression of Tc3A in the worm leads to excision as well as integration of Tc3 .We conclude that Tc3A is an active transposase and is the limiting factor for somatic transposition of Tc3 in Bristol N2 .