Worm Breeder's Gazette 10(3): 28

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.

One Homologous Transformed Line

Andrew Fire and Susan Harrison

Figure 1

It appears that homologous recombination between the injected DNA 
and the chromosome can occur after injection.  This is based on a new 
transformed line, PD125, that was obtained after injecting an inactive 
plasmid carrying most of the unc-54 gene into a paralyzed animal 
bearing an unc-54 null mutation.  The initial plasmid pPD17.09 is 
inactive due to deletions at the 5' end (the promoter region and the 
first four introns including the enhancer are deleted).  The e190 
chromosome has a 400b.  deletion within the myosin coding sequence.  
In general, when pPD17.09 is injected into e190, no rescue is obtained.
Surprisingly, one set of injections yielded a single well rescued 
transformed line, designated PD125.  The homozygous rescued line is 
viable and is phenotypically indistinguishable from wild type.
We have extensively analyzed the DNA of homozygous PD125 animals on 
southern blots.  There is one copy of the unc-54 gene which has the 
restriction map of the recipient chromosome, i.e.  with the 400bp e190 
deletion and no other evident modifications.  The line also has a 
second copy of the unc-54 gene which is a recombinant between incoming 
plasmid DNA and the recipient chromosome.  This copy has an intact 5' 
region of the gene, containing several kb of sequences not present in 
the plasmid pPD17.09 and also carries the 400bp segment deleted in 
e190.  We have confirmed the recombinant nature of this chromosome by 
using several combinations of enzymes that cut both in the upstream 
region deleted in 17.09 and in the internal segment deleted in e190.
The recombinant chromosome contains sequences at the 3' end of the 
gene which diverge from both the parent plasmid and recipient 
chromosome.  The point of divergence is precisely at the end of the 
homology between pPD17.09 and the recipient chromosome.  The 3' 
sequences in the recombinant chromosome have not been identifed but do 
not correspond with any of the sequences in pPD17.09, or to any of the 
sequenced regions around the unc-54 locus.
The structure of the recombinant chromosome is consistent with a 
number of recombination mechanisms.  The site of the 3' divergence 
might suggest that a heteroduplex was formed at some point between 
plasmid and recipient DNA, progressed as far as possible on the 3' 
side (to a terminal 'holiday' structure) and was resolved by complete 
clevage followed by ligation to some unresolved sequence.  Without 
more examples these will remain simply spaghetti-speculation.
Another upshot of all this is that it is conceivable (at a low 
frequency) that a cosmid carrying the site of a mutation but not the 
whole gene could rescue by the same kind of homologous recombination 
event.  We have of course done a lot of 'negative controls' like this 
in the past and never seen this kind of rescuing event, so the 
frequency is likely to be fairly low.

Figure 1