Dissection of Genetic Pathways in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 118

Enhancer Screens Using Null Alleles
Enhancer screens using null alleles (mutations causing complete loss of function
in the corresponding genes) as starting mutations are a widely used and effective way
to isolate functionally redundant genes involved in the same biological process. This
approach was used to study the negative regulation of a Receptor Tyrosine Kinase -
Ras GTPase - Mitogen Activated Protein Kinase signaling cascade (RTK/Ras/
MAPK) mediated by LET-23, an epidermal growth factor receptor (EGFR) like-
typrosine kinase, in vulval indiction in C. elegans ( Fig. 1 ). Activity of LET-23 was
known to be dampened by several functionally redundant negative regulators. A
mutation in any individual regulator is phenotypically silent with regard to vulval
induction but a combination of any two of them displays a hyperinduced-vulval
phenotype due to increased activity of LET-23 ( Sternberg et al., 1994 ). To identify
new LET-23 negative regulators that might be masked by this redundancy,
Hopper et al. (2000) conducted an enhancer screen for the hyperinduced-vulval
phenotype in the background of a null allele of sli-1 (an ortholog of the Cbl family
of ubiquitin ligases), a known negative regulator of LET-23, showing no phenotype
on its own ( Hopper et al., 2000 ). This screen identified a novel negative regulator,
ark-1, which encodes an ortholog of the Ack-related nonreceptor tyrosine kinase.
This gene was later found to be a target of LIN-12/Notch lateral signaling and to
mediate the interaction between LET-23 signaling and LIN-12 pathway during their
cooperative regulation of vulval cell-fate specification ( Yo o et al., 2004 ).
Although an enhancer screen using a null allele is powerful in identifying func-
tionally redundant genes, it has limitations. As a null allele does not produce a
protein to regulate downstream components or be influenced by gene products acting
upstream, the linearity of the signaling relay is interrupted and the activity of the
corresponding pathway is lost. Additional mutations in genes that act either
upstream or downstream would not further enhance the initial phenotype of the null
allele. Thus, an enhancer screen using a null allele is ineffective in identifying
components that act upstream or downstream of the pathway where the starting null
allele resides.
Enhancer Screens Using Hypomorphic Alleles
A hypomorphic mutation causes partial loss of gene function, which leads to a
reduction in the activity of the encoded gene product in the signaling pathway in
which this gene is involved. This reduction can be enhanced in various situations
( Fig. 2 ): (1) loss or reduction in function of a gene that encodes a physical interacting
partner of this hypomorphic allele; (2) loss or reduction in function of another gene
acting in the same or the parallel pathways that functionally compensate for each
other. Thus, an enhancer screen using a hypomorphic mutation can identify a broad
range of interactors that function in either the same physical complex or the same
pathway (upstream or downstream), as well as genes that act in redundant pathways.
For example, in an enhancer screen using a hypomorphic allele of lin-45, a critical