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P-element insertion question

Greg ggloor at julian.uwo.ca
Fri Feb 5 09:54:58 EST 1999


In article <o0ku2.1183$t5.8452712 at WReNphoon2>, jbanders at helix.nih.gov
(John Anderson) wrote:

John,
 
> I know that when a P-element mobilizes, it can excise
> imprecisely, taking away some flanking DNA. My question
> is, when this occurs and then the P-element reinserts
> somewhere else in the genome, does it insert precisely,
> losing the flanking DNA or can it insert the flanking
> DNA into the new site?

Actually, P elements do not excise and take flanking DNA with them. Sved
and Engels showed in a whole series of papers (see references below) that
opposite ends of two different P elements (see Svoboda) could transpose.
The end result is what appears to be a local hop, with an associated
deletion or duplication of flanking DNA (see Gray). Essentially, it
appears as if one end of the P element transposes to a nearby site, and
the other end stays put. This information can be used to design a rational
deletion or duplication strategy (see Preston).  The basic idea is as
follows. Suppose you have a P element inserted near a gene of interest
that you wish to delete into. I'll represent the chromosome as ---- or ==
and your gene as YFG and the P element as <P>. Markers will be M1 and M2,
they do not have to be close to YFG.

------M1--------------<P>-YFG-------------M2---------   chromosome
=======+==================YFG==============+=========   homologue

add transposase and select for recombinant chromosomes that have a
recombinationto the right of YFG. In these chromosomes you will recover
two types of events in which the right P element end has inserted either
to the left of itself, producing a duplication:

------M1--------------<P>==YFG=============+==========

or to the right of itself producing a deletion:

------M1-------------<P>G=============+==========

If the insertion is somewhere in YFG (as shown), then you can pick it up
by a PCR-based screen. You can also repeat the process with the deletion
chromosome and select for recombinants on the other side to effectively
move the P element to the right, delete YFG and reconstitute the remainder
of the chromosome. Note that in this process the P element remains.

A nice description of this is at:

http://www.wisc.edu/genetics/CATG/engels/Pelements/Pt.html#deletions


Gray, Y. H., Tanaka, M. M. and Sved, J. A. (1996) Genetics, 144(4), 1601-10.
Preston, C. R., Sved, J. A. and Engels, W. R. (1996) Genetics, 144(4), 1623-38.
Svoboda, Y. H., Robson, M. K. and Sved, J. A. (1995) Genetics, 139(4), 1601-10.

Good luck!

-- 
Best Regards.....Greg Gloor     | You cannot exploit what you have not |
ggloor at julian.uwo.ca            |              discovered.             |
519-661-3526 fax 519-661-3175   |      Basic Science is important!     |
http://www.biochem.uwo.ca/      |------------ My thoughts -------------|



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