Worm Breeder's Gazette 11(4): 29

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Molecular Cloning of lin-15 by the Candidate Gene Approach

Linda S. Huang, Jane E. Mendel, Phoebe Tzou and Paul W. Sternberg

Figure 1

We have rescued the lin-15 multivulva.(Muv; visible or AB) and 
synthetic (syn; class A and class B) Muv phenotypes with cosmid 
PS#74B3.  This cosmid maps to the far left (formerly, far right) end 
of the clb-1 contig on the X chromosome and was originally isolated 
because it weakly hybridizes to the G-protein PCR clone 1, as reported 
in WBG11(2):32.  As we had been trying to clone lin-15 (see WBG11(3)
:60 for last update), we knew from the map data that lin-15 should lie 
either at the right edge of the clb-1 contig or in the gap off the end.
Since PS#74B3 appeared to map closely to lin-15 on the physical map 
and because the putative G-protein was a candidate gene for a genetic 
locus presumably involved in signal transduction, we decided to 
microinject the cosmid to test whether it rescued lin-15.  As we had 
hoped, PS#74B3 rescued lin-15, although the G-protein hybridizing 
region did not rescue.  A 'reverse northern' analysis identified a 6.8 
kb BamHI fragment which lay within the region defined as necessary for 
lin-15 function by injection.  When the 6.8 kb fragment was used to 
screen Stuart Kim and Chris Martin's cDNA libraries, we identified two 
classes of non-cross hybridizing cDNAs which mapped to opposite sides 
of our 6.8 kb fragment, with cDNA2 apparently more abundant than cDNA1.

[See Figure 1]
Subsequent injection results showed that genomic DNA containing 
cDNA1 but not cDNA2 was sufficient to rescue the Muv phenotype.  Also, 
RFLP analysis picked up a small deletion in the lin-15(n767) class A 
allele in the region of cDNA1.  DNA which does not contain cDNA1 but 
does contain cDNA2 did not rescue the lin-15(n765) Muv phenotype (n765 
is Muv at >20 C, class B at 15 C)--however, an RFLP was found in n765 
to the right of cDNA2.  Also, genomic DNA which contains both cDNA1 
and cDNA2 rescues the Muv phenotype much better than DNA containing 
only cDNA1.  Furthermore, all of the four EMS induced visible Muv 
alleles plus the three lin-15 alleles generated by Stuart Kim in the 
TR679 mutator strain contain large deletions of DNA in this genomic 
region, with n377 deleting the 6.8 kb region entirely.  We believe 
that the lin-15 locus contains at least two independently mutable 
regions such that a hit in one causes a class A syn Muv phenotype 
while a hit in the other causes a class B syn Muv phenotype, the 'two 
hit model'.  Single hits in both regions or a large deletion covering 
both regions are necessary for a visible Muv phenotype.  This model is 
supported by the facts that: 1. the four EMS induced visible Muvs 
contain large deletions even though the frequency of EMS induced 
deletions is believed to be no greater than 10%, suggesting a strong 
selection for deletions in lin-15 visible Muv hunts.  2. we were 
unable to recover a null allele in a non-complementation screen of 35,
000 F1 worms using EMS (see WBG11(3):60 for details), suggesting that 
single point hits can not cause a null.  3. DNA covering the n765 
polymorphism is not sufficient to rescue the Muv phenotype, suggesting 
there may be a second hit elsewhere in the region.  4. although it 
doesn't seem possible to obtain visible Muv mutations as EMS single 
point hits, visible lin-15 alleles are easily obtained in F2 screens 
for syn Muvs starting with an opposite class syn Muv allele.  A 
visible lin-15 allele recovered in a syn Muv screen using n767 only 
shows the deletion originally found in n767, suggesting there is a 
second point hit elsewhere in the region.  It is possible that cDNA1 
and cDNA2 are differentially spliced 3' exons of a common 5' end, or 
that cDNA1 and cDNA2 are driven by the same control region.  Another 
possibility is that the lin-15 locus could comprise two closely linked 
genes, one which is a class A syn Muv and the other which is a class B 
syn Muv.  We are working to resolve these issues and also to further 
elucidate how lin-15 functions in the vulval development pathway by 
further characterization of the cDNA clones and the associated genomic 
DNA surrounding those regions.

Figure 1