Worm Breeder's Gazette 12(5): 22 (February 1, 1993)
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.
We have previously reported the intron-exon structure of the
putative transposase gene Tc1A )of the transposable element Tc1
and the binding of Tc1A to the inverted repeat of Tc1 (WBG 12 (3),
pg. 87). In a manner similar to Tc3 (see WBG 12(4), pg. 15), we
analyzed somatic transposition of endogenous Tc1 elements in
transgenic N2 animals in which we induced Tc1 A expression. In non-
transgenic, or in non-induced transgenic N2 ,we found approximately
3 Tc1 insertions in the gpa-2 gene per 1 ug of worm DNA
(equivalent to approximately 10.000 genomes). A 15 fold increase in
the number of Tc1 insertions is seen after Tc1 A expression in the
transgenic strain. Therefore, we conclude that the Tc1A gene
product is a transposase and that it is a limiting factor for
transposition in N2 . Tc1 A induction does not result in Tc3
transposition, which requires expression of its own Tc3
transposase. We expressed Tc1A in E. coli and could show the
appearance of a polypeptide which binds specifically to the inverted
repeat of Tc1 .We have characterized the DNA binding further by
DNase I footprinting and observed protection between basepairs 3
and 30 relative to the end of the transposon. This suggested that the
terminal 6 nucleotides shared between most members of the Tc1
family (TACAGT) are not involved in sequence specific binding of the
transposase to the ends of the element. Mutation analysis of the
inverted repeat showed that the TAGATC sequence is indeed not
important for bindinq of Tc1A to the inverted repeat. Perhaps they
play a role in a later step in the transposition pathway. Purification
of the polypeptide used for these assays revealed that a (probably
proteolytic) derivative of Tc1A was responsible for the high-
affinity DNA binding activity. In contrast, the full-length Tc1A
protein has a very low affinity for the inverted repeat, which
suggests the presence of a domain in Tc1 A which regulates the DNA
binding potential. A similar result was obtained when we analyzed
Tc1 A DNA binding activity in nuclear extracts prepared from
induced transgenic animals. In a gel retardation assay we detected a
complex which migrates at a position expected for a smaller version
of Tc1A .We do not know what the significance of this observation
is, but one may speculate about the occurrence of a smaller version
of Tc1A which acts as a repressor in vivo. Analysis of carboxy-
terminal deletion mutants of Tc1A showed that the domain required
for site-specific binding is contained within the first 63 amino
acids. Thus, the coding information for inverted repeat binding is
mostly contained within the first exon of Tc1A .Furthermore, there
is a second DNA binding domain, because a construct in which the
first 70 amino acids of Tc1 A are absent, was previously shown to
contain a strong general DNA binding domain (Schukkink and
Plasterk, NAR 1990). In summary, we conclude that Tc1A is a
transposase and has two independent DNA binding domains, one N-
terminal for the binding to the inverted repeat of Tc1 and a second
non-specific DNA binding domain which possibly interacts with
target DNA in the transposition reaction.