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screening scale. As suppressors isolated in the primary screen may not all be specific
to loss of rpm-1 activity, a secondary screen at the microscope level was then
conducted to identify rpm-1-specific suppressors that restored synaptic morphology
using a synapse fluorescent marker. The highlight from this screen was the identi-
fication of three MAP kinases, dlk-1 (MAPKKK), mkk-4 (MAPKK), and pmk-3
(p38-like MAPK), which were found to form a previously uncharacterized p38 MAP
kinase cascade that is negatively regulated by RPM-1 during synapse formation. The
success of this screen speaks to the specificity and utility of a sequential screening
strategy when designed appropriately.
8. Evaluation and Limitations of Forward Genetic Screens
Regardless of various screening designs, a high-quality screen should be able to
identify many or most of the nonredundant components of a biological pathway of
interest. A key question that then arises is how to determine the degree of saturation
(all genes that can be mutated to display a specific phenotype) that a screen reaches.
Empirically, if a screen is saturated, (1) mutations in the same gene, particularly in
small-size genes that usually are less frequently hit, will be repeatedly isolated. This
will be reflected by the fact that multiple alleles fail to complement each other in a
complementation test, which is a genetic experiment to determine if two alleles
reside in the same gene by comparing the phenotype of transheterozygotes of these
two alleles with that of homozygotes for each allele. If the phenotypes of transhe-
terozygotes and homozygotes are the same, it indicates that the two alleles fail to
complement each other, suggesting that the two alleles likely correspond to the same
gene. Conversely, if a screen is not saturated, it is common to see that each mutation
defines a distinct locus. For example, in the lin-35/Rb synthetic lethality screen
described in Section II.A.4, Fay and his colleagues recovered seven mutations
defining seven distinct loci, indicating the screen was not saturated. (2) Unusual
hypomorphic alleles of lethal genes will be identified that normally are less likely to
be recovered than null alleles because these alleles require changes in specific amino
acids. Besides qualitative judgment, the degree of saturation can also be analyzed in
a quantitative manner. A statistic method using Bayesian and maximum-likelihood
calculation may be used to estimate the number of alleles that remain to be found
(
Pollock and Larkin, 2004
).
Many components of genetic pathways have been identified through mutagenesis-
based forward genetic screens. However, as an experimental approach, forward
genetic screens have intrinsic weaknesses in identifying some pathway components.
Even though a forward genetic screen can be performed at a large scale, many genes
may still be missed or rarely hit for several reasons: (1) small-size genes may be
missed because they are too small to be effective targets for mutagenesis; (2) genes
with pleiotropic functions might not be identified because they preferentially give a
phenotype that masks their other functions (
Jorgensen and Mango, 2002
); (3) genes
that when mutated confer early lethality often prevent identification of their later
functions; (4) functional redundant genes that ensure robustness and plasticity to
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