is there transposase repressor construct?

[Kevin Cook-5]

Hi Olaf--

Using a repressor construct in crosses to make deletions from
trans-heterozygous P elements is a reasonable idea. I considered it when
we were using the method to make deletions for the Bloomington Stock
Center collection (described in Parks et al. (2004) Nature Genetics 36:
288-292), but decided it wasn’t worth it for our screens. Let me see if
I can explain my thinking.

First, it’s impossible to predict the exact positions of breakpoints of
deletions made by the Hybrid Element Insertion (HEI) mechanism in the
presence of trans-heterozygous P insertions. One breakpoint should
correspond to the insertion site of one of the starting insertions. The
other breakpoint will usually be in the vicinity of the insertion site
of the second P element, but you can’t predict exactly where it will
be--it could be proximal or distal to the second insertion or within the
transposon. No one knows why the hybrid element usually inserts near one
of the starting insertions. It’s probably a physical constraint on the
chromatids involved, but that’s just speculation. For an in-depth look
at the HEI mechanism, you should work your way through the following
three papers. It’s straightforward to see how their results apply to
trans-heterozygous P insertions.

Gray, Y.H., Tanaka, M.M., Sved, J.A. (1996). P-element-induced
recombination in Drosophila melanogaster: hybrid element insertion.
Genetics 144(4): 1601--1610.
Preston, C.R., Engels, W.R. (1996). P-element-induced male recombination
and gene conversion in Drosophila. Genetics 144(4): 1611--1622.
Preston, C.R., Sved, J.A., Engels, W.R. (1996). Flanking duplications
and deletions associated with P-induced male recombination in
Drosophila. Genetics 144(4): 1623--1638.

We were generating very large deletions. A local hop in the intermediate
generation preceding the Hybrid Element Insertion event giving rise to
the deletion wouldn’t change the predicted size of the deletion by very
much. Indeed, most of our deletions had the expected breakpoints at the
level of polytene cytology. A few kb of doubt about the positions of the
endpoints probably doesn’t matter too much for a deletion in the
megabase size range. I figured that anyone wanting to know the exact
genomic coordinates of the deletion breakpoints could characterize them
by sequencing off the ends of the P element retained on the chromosome
following the HEI deletion event.

I understand that it’s a different situation when you’re trying to
generate small deletions. A few kb difference between the size of the
deletion based on the insertion sites of the starting P elements and the
actual deletion size can make a big difference if you’re trying to
delete specific genes. Using a repressor construct MIGHT make sense, but
I think you still need to think about whether it’s worth the effort.
Basically, you’re playing a numbers game. Not every P element hops in
the presence of transposase in every generation. The probability of P
transposition in the intermediate generation of the screen is not that
high. It can happen, but the odds are in favor of it not transposing.
You may save yourself work by doing the screens as we described in Parks
et al. and then characterizing the breakpoints in the resulting
deletions. You can keep the ones with desirable breakpoints and discard
the others.

It would be interesting to know if the inclusion of a repressor
construct would prevent transposition in the intermediate generation of
the screen, so I’m not discouraging you from trying. I’m just saying
that it’s not absolutely necessary to get what you want.

If you want a single gene deletion, you may be able to get it more
easily from a conventional male recombination screen employing a single
P insertion as described in the Preston, Sved and Engels article cited
above. Those screens are easier and generate a lot of deletions of less
than ~10 kb. I don’t think screens using trans-heterozygous P insertions
would be my first choice for recovering small deletions. Of course, the
FLP-FRT system would be my first choice, but I’m guessing that
appropriate insertions aren’t available for the gene you’re interested
in if you’re thinking about HEI screens. You could also check to see if
appropriate insertions are available to use hobo transposition to make
deletions using P{wHy} insertions (see
http://flystocks.bio.indiana.edu/Browse/insertions/PwHy-top.htm).

I hope this helps a bit,
Kevin

--
Kevin Cook, Ph.D.              
Bloomington Drosophila Stock Center
Department of Biology          
Indiana University              
1001 E. Third St.              
Bloomington, IN  47405-7005  

kercook@...
812-856-1213 (office), 812-855-2577 (fax)
http://flystocks.bio.indiana.edu

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