Biology Reference
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determined by the genotype of the mother; new-1(*) could be either domi-
nant or recessive, since the mother is homozygous). An alternative possibil-
ity is that the cross was unsuccessful and the new-1(*) stock has a Him (high-
incidence males) phenotype; this can be resolved simply by inspecting the
original new-1(*) stock for males and then repeating the cross, using purged
hermaphrodites if necessary (see below).
In cases where new-1(*) appears to be dominant and/or has a maternal effect,
the mapping cross will need to be repeated using purged hermaphrodites.
To obtain purged hermaphrodites, pick
20 young adult New hermaphro-
dites onto a seeded plate and incubate at 20
. After 48 h, transfer the adult
worms to a fresh plate and inspect them to see if any have expended their
sperm. Early-stage purged hermaphrodites will have unfertilized oocytes in
the uterus. Unlike fertilized eggs, these lack an eggshell and give the uterus a
fairly homogeneous, light brown appearance. As the hermaphrodites run out
of sperm, they will begin to lay unfertilized oocytes on the plate. These
superficially resemble fertilized eggs; however, they are soft and easily
ruptured, typically degenerating into irregular brown blobs within the E. coli
lawn. Late-stage purged hermaphrodites will have an empty uterus and the
oocytes within the proximal oviduct will be compressed to give a ''piano
key'' appearance. If you are uncertain, it is better to pick slightly later-stage
purged hermaphrodites; however, if they are too old they might not produce
progeny in response to mating.
Set up the mapping cross using 10-20 young adult CB4856 males and 3-10
purged New hermaphrodites. Incubate at 20
and inspect after 24 h. If no
eggs are present on the plate, then the hermaphrodites were too old and the
cross needs to be repeated. If eggs are present, then incubate at 20
for an
additional 48 h, then inspect the F1 progeny. If the hermaphrodites were fully
purged, then
50% of the progeny should be males.
Clone 10 young adult F1 hermaphrodites. Incubate at 20
for 24 h, then
transfer each to a new plate, and continue incubation at 20
. Inspect the F2
progeny 4 days after the hermaphrodites were initially cloned (assuming that
New is optimally scorable in adults).
b) Dominant Zygotic Effect
If substantially more than 25% (and up to 75%) of the F2s are New, this is
consistent with a dominant/semi-dominant, zygotic-effect mutation. Pick New
and non-New F2s for SNP mapping, as described in Section VI.
A dominant maternal effect mutation that has incomplete penetrance could
also produce this ratio, and this should be considered if the SNP mapping
procedure does not provide evidence of linkage. Note that such results are also
consistent with the unlikely possibility that the New phenotype is due to a
mutation in the mitochondrial genome. If desired, this possibility can be tested
by determining whether
new-1(*)
can be transmitted through males (see
Tsang and Lemire, 2002
).
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