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RTK-Ras-MAPK component encoding a Raf protein, Rocheleau et al. (2002) iden-
tified novel alleles of known components of this pathway that function either
upstream or downstream of lin-45, including sem-5, sos-1, lin-1, and ksr-1, and
alleles in two new components, eor-1 and eor-2, likely acting downstream of or in
parallel to mpk-1 ( Fig. 1 ). The spectrum of genes identified highlights the efficiency
of using hypomorphic alleles in enhancer screens.
Another example showing the usage of hypomorphic alleles in identifying genes
with redundant functions comes from a screen carried out by Schwabiuk et al.
(2009) . They discovered a novel function for a gene, sdn-1, in regulating migration
of the distal tip cells (DTC) that lead gonad extension. sdn-1, which encodes an
ortholog of a type I transmembrane proteoglycan syndecan-2, was previously impli-
cated in axon guidance ( Rhiner et al., 2005 ) and in epidermal enclosure
( Hudson et al., 2006 ). Its new functional role in DTC migration was found in a
screen for mutations that enhanced the DTC migration defects caused by a hypo-
morphic allele of unc-5, which encodes a receptor for the axon guidance cue, netrin.
sdn-1 would not otherwise have been identified in simple screens using wild-type
worms or enhancer screens using a null allele of unc-5, because the ensuing func-
tional characterization revealed that all sdn-1 alleles (null and hypomorphic) were
phenotypically silent in DTC migration on their own and importantly no enhance-
ment was observed in the double mutants between an unc-5 null allele and any of
sdn-1 alleles, indicating that sdn-1 function is linked to unc-5 activity.
For some enhancer screens, temperature-sensitive alleles (''hypomorphic'' equiva-
lents) can also be used. These alleles often display incomplete phenotypic penetrance at
intermediate temperature between restrictive and permissive temperatures. Additional
mutations that increase original penetrance of phenotypes at intermediate temperature
are isolated and cloned. This temperature strategy has been used in a number of
enhancer screens, such as a screen to identify genes involved in the neddylation process
( Dorfman et al., 2009 ), as well as LIN-12/Notch signaling ( Qiao et al., 1995 ).
Synthetic Lethality Screen Using Extrachromosomal Arrays
In some enhancer screens, the combination of two mutations can cause synthetic
lethality: the disruption of a single gene displays no discernable or a very subtle
phenotype, whereas simultaneous disruption of two or more genes causes lethality.
Synthetic lethality poses a challenge for mutation recovery as it is usually unknown
whether a starting mutation has synthetic lethal partners. One way to recover syn-
thetic lethal mutations is to devise an enhancer screen using a starting mutant
carrying an extrachromosomal array that rescues potential lethality ( Fig. 3 ). These
arrays are transgenes introduced into worms by gonad microinjection ( Mello and
Fire, 1995 ). Due to their extrachromosomal nature, these transgenes are stochasti-
cally lost during meiosis and mitosis, and are only expressed in the progeny or
daughter cells containing them. For enhancer screens, the transgenes carried by
the starting mutant are often designed to express two proteins simultaneously:
(1) a wild-type copy of a starting-mutation-harboring gene for avoiding synthetic
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