Worm Breeder's Gazette 9(1): 31

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Progress in cloning the mec-3 gene

J. Way and M. Chalfie

Figure 1

We used the spontaneous mutant u298 isolated from TR679 to clone at 
least part of the mec-3 gene by the now-standard 'transposon tagging' 
method used by Don Moerman to clone unc-22 and Iva Greenwald to clone 
lin-12.  The original mutant was backcrossed to N2 several times, then 
crossed with unc-24 (0.7 m.u.  to the left) and dov-20 (0.3 m.u.  to 
the right).  We identified a single Tc1-containing EcoRI fragment 
which always co-segregated with the mec-3 mutation.
From an outcrossed strain, we cloned a Pst-Bam fragment containing 
the Tc1 insertion into the plasmid pUC8.  We also constructed a 
restriction map of this region of the C.  elegans genome.
[See Figure 1]
A plasmid derivative in which the Tc1 had been excised (pTU1) was 
used to probe Southern blots of various mutants.  All mec-3::Tc1 
insertions are at the same site in the same orientation.  (As 
described above, we may only have two independent insertions.) TR679 
and a revertant of one of the insertions have the same pattern as N2 
for all enzymes tested.  The Tc1 elements are identical to the 
sequenced Tc1 (Rosensweig et al.  NAR 11, 4201) in their restriction 
map as far as we know.  (The cloned Tc1 does not have a HindIII site; 
we don't know about the others.) We can see a faint somatic excision 
band at the wild-type position in digests of each Tc1 mutant.  EMS 
induced mutations show no change in pattern.
We have not yet identified a transcript from this region; nor have 
John Sulston and Alan Coulson identified a cosmid contig containing 
our clone (because the cloned DNA is too small).  We are doing all the 
obvious things.
Information about the genetic map around mec-3Positions of cosmids.  
Two cosmids which pick up N2/BO RFLPs were positioned relative to the 
markers unc-24, mec-3 and dpy-20.[See Figure 2]
All 4/4 + mec-3 recombinants derived from eDF18 / + 
+ (BO) heterozygotes had the BO pattern when probed with BO555, so 
this cosmid maps near or to the left of the right endpoint of eDF18.
The CGBP1 cosmid was shown by Don Moerman to contain a hotspot for 
spontaneous Tc1 insertion.  When used as a probe, this cosmid reveals 
a PstI polymorphism (the polymorphic bands are both >20 kb), which 
maps as shown, and a cross-hybridizing PstI polymorphic region which 
maps to the left of unc-24.A third cosmid, BO5I5, sent to us from 
David Baillie, maps to the right of mec-3.  We have not positioned it 
relative to dpy-20.TR679.  When either of the above cosmids is used to 
examine u298 strains containing this region of chr.  IV from TR679, 
the Bristol pattern is revealed.  However, in the course of 
outcrossing the mec-3::Tc1 insertion described above, it was apparent 
that there are at least 4 additional Tc1 elements between unc-24 and 
dpy-20 in this particular TR679 stock.  All of these Tc1s are in 
different sized EcoRI fragments from those in analogously constructed 
BO/N2 hybrids containing BO DNA around mec-3.

Figure 1