Worm Breeder's Gazette 11(2): 79

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Psoralen Mutagenesis Produces Deletions at High Frequency

L. Edgar, M. Perry, M. Yandell, P. Newmark and W.B. Wood

Figure 1

We are continuing characterization of unc-22 mutations caused by 
trimethyl-psoralen (TMP) as part of an effort to determine whether 
this mutagen produces a different mutational spectrum than EMS.  TMP, 
activated by near-UV light, cross-links DNA, so we might expect a high 
frequency of rearrangements and deletions as a consequence of breakage 
and repair.  Preliminary analysis of 22 homozygous-viable unc-22 
mutations by Southern blot gave the following 
distribution:
Deletions: 
8
Possible Inserts: 
1
No Detectable Polymorphism: 
12
The probes used were DM18 and DM20, which cover most of the 
transcribed region and 10kb of 5' upstream sequence.  (DNA probes 
kindly provided by Carol Trent.) Sizes of the 5 internal deletions 
ranged from 100 bp to 3.5 kb; the other three represent a 5' and two 
3' end fragment deletions.  One of these (5') appears >10 kb.  We plan 
to extend this analysis using cosmid probes to determine whether any 
of the mutations showing no polymorphism represent deletions of 
sequences not covered by the probes used so far.  Mutagenesis of L4's (
60' light at 300  w/cm2 following a 15 min TMP soak) gave a mutation 
rate of approximately 10+E-4, comparable to EMS rates for unc-22.  In 
addition to looking for homozygous mutants, we screened F2 plates for 
heterozygous twitchers representing homozygous inviable mutations, 
suspecting that large deletions in the unc-22 region might include 
nearby essential genes.  We retrieved 9 such mutants in a run 
producing 15 homozygous viable mutants.  Several of the former are 
currently being balanced for analysis.  None extend to dpy-20.  Thus 
TMP appears quite efficient at generating small deficiencies, which 
may make it a useful mutagen for producing null alleles and for 
molecular tagging.  Protocol available; we would be interested to know 
if it works on your favorite gene.
[See Figure 1]

Figure 1