Biology Reference
In-Depth Information
II. Strategies for Identifying Components in
Pathways of Interest
Three general strategies are currently used by C. elegans biologists to discover the
specific components of pathways: (1) forward genetic screens that encompass a
diverse array of screening methods and can be complemented with reverse genetic
approaches; (2) functional genomic approaches that utilize genome-wide analysis of
transcription, protein-protein interaction, DNA-binding site analysis, and loss-of-
function techniques; and (3) systems biology approaches that integrate the use of
functional genomic techniques.
A. Forward Genetic Screens
1. Forward Genetics and Reverse Genetics
Biological processes are precisely regulated by highly coordinated gene regula-
tory networks that are comprised of numerous interacting signaling pathways. The
basic constituent of signaling pathways are proteins encoded by corresponding
genes. Perturbation of these genes could cause deregulation of associated pathways
or even the entire network. Deregulation sufficient to disrupt a biological function
may result in an observable outcome referred to as a phenotype. The collective status
of perturbed genes is called genotype. The biological relationship in which a geno-
type determines a phenotype is the foundation for dissecting genetic pathways.
Forward genetic approaches (investigation directed from phenotype to genotype)
and reverse genetic approaches (investigation directed from genotype to phenotype)
are two powerful ways of elucidating the function of genes that regulate a biological
process of interest. Forward genetic screens identify genes in an unbiased manner
based on phenotypes of mutants. The screens start by searching for a desired
phenotype caused by a mutation that is introduced into a gene by mutagens, such
as EMS (1-methylsulfonyloxyethane, also known as ethyl methanesulfonate) or
ENU (1-ethyl-1-nitrosourea, also known as N-ethyl-N-nitrosourea). Identity of the
mutation-harboring genes can be determined by positional cloning or candidate-
gene testing. Forward genetic studies in C. elegans have made significant contribu-
tions to our understanding of a wide range of developmental processes. For example,
the forward genetic screens pioneered by Nobel Prize Laureate Robert Horvitz for
mutants defective in programmed cell death (PCD) identified the underlying genetic
pathways that direct apoptosis, a process conserved among metazoans, including
humans (
Metzstein et al., 1998
).
Similarly, reverse genetics has provided considerable insights into many biolog-
ical processes. Reverse genetic approaches begin with a set of genes with known
sequences that are of particular interest such as disease-related genes (
Ahringer,
1997; Barr et al., 2001; Derry et al., 2001
). Genes are inactivated by target-selected
approaches such as creation of deletion mutants using chemical mutagens or UV
light (
Gengyo-Ando and Mitani, 2000; Jansen
et al., 1997; Liu
et al., 1999
),
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