Worm Breeder's Gazette 14(1): 24 (October 1, 1995)

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.

Tc1 jumps in vitro

Chris Vos, Ronald Plasterk

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

Over the last few years, many details about the mechanism of Tc1 and
Tc3 transposition have been obtained by studying the process in vivo.
Jumping is catalysed by the transposase protein encoded by the
element. It generates double strand breaks at the ends of the
transposon; this results in a linear excised element with 2 bp
staggered 3' overhangs (Cell 79:293-301, 1994). Integration occurs
always into a TA dinucleotide. Notably, Tc1 and Tc3 appear to have
quite distinctive integration patterns in vivo as shown for a randomly
chosen gene area, i.e. a fragment of the gpa-2 gene (NAR 22:
5548-5554, 1994).

We have now developed a Tc1 in vitro transposition assay. We make use of
a nuclear extract derived from a stable transgenic worm strain with a
transposase gene under the control of a heat shock promoter (Genes & Dev.
7 ; 1244-1253, 1993). A donor plasmid with a marked Tc1 element is
preincubated with such an extract, and subsequently a target plasmid is
added. After the reaction, products were analyzed in both a physical and
a genetic assay. In vitro reaction products were directly visualized by
Southern blotting and by primer extension. Double strand breaks at one
transposon end or at both transposon ends are detectable. The mapping
of the ends of the excised element confirms the presence of the 2 bp
3' overhang as previously determined for Tc3 in vivo. Double strand
breaks at transposon ends can occur on either a supercoiled or a
linear substrate and with substrates containing a single transposon
end. Finally, addition of ATP or GTP does not influence the efficiency
of Tc1 excision.

Experiments to determine the cis-requirements for Tc1 excision in vitro
show that only the terminal 26 bp of Tc1 are sufficient. These sequences
contain the transposase binding site.  Mutation of the transposase binding
site abolishes excision. Also, mutation of the terminal 4 base pairs of
Tc1, as well as mutation of the flanking TA sequence, which are conserved
between Tc1, Tc3 and many other members of the Tc1-family, are detrimental
for excision in vitro.

In the genetic screen to detect actual integration products, the reaction
mix is electroporated into an E. coli strain which allows selection for
the Tc1-borne antibiotic resistance and against the donor plasmid.
Integration products are obtained which faithfully reflect in vivo
transposition events: Tc1 is integrated into a TA dinucleotide, which is
duplicated. Among the 80 sequenced independent Tc1 insertions, two
exceptions were found where Tc1 had integrated at the sequence TTG or CCT.

We included a 1.4 kb gpa-2 sequence in the target plasmid to be able to
compare the Tc1 in vitro integration pattern with the one previously
determined in vivo. Therefore, 62 independent in vitro integration events
in gpa-2 were sequenced. Itappears that hot sites in vivo are hot in vitro
and that cold sites in vitro are cold in vivo. Therefore, the integration
pattern is not dependent on chromatin structure or the transcriptional
status, but rather reflects site-specificity of the transposition complex.

The presence of Tc1 related elements in widely divergent phyla has raised
the hypothesis that transposition does not require species-specific host
factors in addition to the element encoded transposase.  Therefore, we
expressed transposase in a heterologous system using recombinant baculo
virus. Total cell lysates from infected cells were proficient for Tc1
transposition. This shows that no C. elegans specific protein is required
besides Tc1 transposase. The next step will be to determine whether
purified transposase is sufficient for the whole transposition process or
if general host factors are required.