Mapping Mutations in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 13

permissive temperature. These crosses were done at permissive temperature and F1
cross progeny were identified based on their non-Unc phenotype. F1 hermaphrodites
were then selfed at restrictive temperature to obtain information regarding linkage
and dominance of the modifier mutations.
Approximately half of the modifier mutations behaved as expected for single gene
dominant alleles, with > 50% of the F2 progeny exhibiting fertility. Some of these
appeared to be intragenic revertants in gon-2, since all of the Unc progeny were
fertile, and this was confirmed by DNA sequencing (E.J.L., unpublished). Most of
the dominant mutations were clearly unlinked to gon-2, since 25% of the Unc
progeny were sterile. Due to their dominant nature, these mutations could not be
tested for allelism via complementation testing. However, we deduced that each was
X-linked, because when we crossed F1 males (genotype gon-2(q388); mod/0) with
hermaphrodites of genotype gon-2(q388) unc-29(e1072) and raised the progeny at
the restrictive temperature, nearly all of the non-Unc hermaphrodite progeny were
fertile, but nearly all of the male progeny were gonadless; through positional cloning
and sequencing we subsequently determined that all were alleles of the same gene,
gem-1 (gon-2 extragenic modifier) ( Kemp et al., 2009 ).
Among the recessive mutations, a few were clearly linked to unc-29, since all of
the Unc F2 progeny were fertile. These were confirmed to be intragenic revertants of
gon-2 by DNA sequencing. The other Mod strains segregated 25% fertile F2
progeny, consistent with a single gene recessive mutation. We tested these for
linkage to him-8 by cloning fertile F2s and scoring the Him phenotype (i.e., exam-
ining their broods for the presence of high frequency males). In two cases, approx-
imately 25% of the fertile F2s were homozygous for him-8(e1489), indicating a lack
of linkage. We subsequently used conventional mapping methods to assign these
mutations to chromosomes II (gem-2) and III (gem-3) (E.J.L., unpublished). In the
other cases, few if any of the fertile F2s were Him, suggesting that the mutations were
balanced by him-8(e1489) on chromosome IV. These mutations failed to comple-
ment each other, and we used positional cloning and sequencing to assign them to the
same gene, gem-4 ( Church and Lambie, 2003 ).
D. General Strategies for Modifier Mutations
Essentially the same methods that we used for characterizing suppressors of
gon-2 can be applied for the initial characterization of any Mod mutations. In the
case of extragenic Mod mutations, one would then apply the following strategy
(e.g., with prim-1 tightly linked to unc-29). Cross hermaphrodites of genotype
prim-1(*) unc-29(e1072); mod-1(*) with CB4856 males. Pick one non-Unc F1
hermaphrodite onto each of 10 plates. For a zygotic-effect mutation, proceed with
SNP mapping as in Section VI by picking Mod and non-Mod F2s. For maternal
effect mutations, pick 100 F2s onto each of two plates, allow them to lay eggs for
1-2 h, and then rinse off with M9 buffer to remove adults. Pick Mod and non-
Mod F3s for SNP mapping.