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In addition, selection screens can be conducted using temperature sensitive (ts)
lethal mutants. Growing such mutants at restrictive temperature usually induces
embryonic or larval lethality. In screens for suppressors of lethality of ts alleles,
mutants that restore the viability of embryos or larvae can be easily identified. For
instance, PAR (partitioning defective) proteins, first identified in forward genetic
screens in C. elegans, are highly conserved regulators of cell polarity and asymmet-
ric cell division (
Kemphues et al., 1988
). To gain insight into the precise mechanisms
by which PAR-1, a Ser/Thr kinase, regulates embryonic asymmetric cell division,
Spilker et al. (2009)
performed a genome-wide RNAi screen on a temperature
sensitive par-1 allele and identified several genes that when their activity is reduced
specifically suppress the embryonic lethality of par-1. One of the identified sup-
pressors was mpk-1, which encodes a mitogen-activated protein (MAP) kinase.
Reduced activity of mpk-1 restored the asymmetric distribution of cell-fate specifi-
cation markers in par-1 mutants. In addition, disrupting the function of other com-
ponents of the MAPK signaling pathway also suppressed par-1 embryonic lethality.
These results revealed that MAP kinase signaling is involved in antagonizing PAR-1
activity during early C. elegans embryonic polarization.
7. Sequential Screens
The specific phenotypes of a biological process of interest may not always be
suitable for large-scale forward genetic screens. One common reason is the difficulty
in observing the phenotype. To make genetic screens applicable for such a biological
process, it is often possible to score a more readily detected phenotype, such as
lethality or uncoordinated movement (primary screen), that allows isolation of a
broader scope of mutations including those specific for the process of interest. The
specific mutations are then identified through a secondary screen. A good example
using a sequential strategy was a screen aimed at identifying genes involved in
regulation of presynaptic terminal formation. This was accomplished by seeking
suppressors of a mutation in the RING finger/E3 ubiquitin ligase gene rpm-1, a key
regulator of synapse formation (
Nakata et al., 2005
). Mutations in rpm-1 result in a
disorganized presynaptic structure but they cause no defects in locomotion.
Although this phenotype can be observed with fluorescent synaptic markers, its
subcellular microscopic-level nature limits the screening scale. To facilitate the
identification of genes interacting with rpm-1, they devised a sequential screen
for suppressors of an rpm-1 mutation. In the primary screen, instead of screening
solely in the rpm-1 mutant background, they screened for suppressors of a set of
easily scored phenotypes, severe defects in locomotion, and reduction in body size,
caused by introducing (with rpm-1) a mutation in a synaptogenesis gene, syd-1. The
SYD-1 protein regulates the distribution of presynaptic components and when
mutated leads to mild defects in locomotion and egg-laying (Egl) behavior.
Specific suppressors of rpm-1 could be identified by restoration of locomotion
and body size but not the Egl phenotype of the syd-1 mutation. The alternative easily
scored phenotypes used for suppression in the primary screen allowed a large
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