Worm Breeder's Gazette 11(5): 46

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Seeking Trans- and Cis- Regulators of Transposon Tc1

King-Lau Chow and Scott W. Emmons

Figure 1

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  Unpublished data from our 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 
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]

Figure 1