Biology Reference
In-Depth Information
culture), and so chemical genomics has yet to make
a similar impact on systems-level understanding of worm
biology.
Despite the difficulty of delivering many drugs effi-
ciently and at effective concentrations into the worm,
some pioneering work in small molecule screening has
been carried out. Small molecule screens of several
thousand commercially available compounds
[55]
have
identified novel Ca-channel antagonists and a novel
chemical inhibitor of DAF-9 a cytochrome P450 with
a key role in dauer formation
[56,57]
. In addition to
identifying drugs that affect the worm to produce biolog-
ically interesting phenotypes, through the use of down-
stream forward genetics screens, they were often able to
identify the true target of the identified compound, taking
the process of small molecule from lead compound to drug
target in a rapid manner. Crucially, they also developed
a set of rules that are predictive of the uptake and bioac-
tivity of small compounds into worms
[54]
. This should
open the way for more large-scale screening of pre-
selected small molecule libraries, with high coverage of
the chemical space that is predicted to be bioactive in
worms. To date, however, these approaches are in their
infancy, and while such screens are exciting possible
sources both of novel anthelmintics and of key research
tools such as pathway agonists and antagonists, this is
currently a work in progress rather than a fully established
area of systems research in the worm.
The publication of the genome sequence of the worm in
1998 opened the possibility of systematic reverse genetics:
here are all the genes, what does each one do in vivo? To
begin to answer this requires a means of perturbing each
gene in a specific and targetedmanner that can be carried out
at sufficient throughput to analyze an entire genome. In
yeast, the relative ease of chromosome engineering made
the direction obvious: delete the genes one by one by tar-
geted disruption and see what happens. In the worm, no such
technology existed
targeted knockouts were a pipedream.
By pure chance, the key enabling technology for systematic
reverse genetics in the worm was discovered at a time that
coincided nearly perfectly with the assembly of the genome
sequence. The first paper reporting the potent and specific
action of RNA interference (RNAi) was published in 1998
[62]
; by 2000 around 25% of worm genes had been targeted
by RNAi and the effects reported
[63,64]
; and by 2003 the
results of the first genome-scale RNAi screens were pub-
lished
[65
e
69]
. The speed with which RNAi was adopted
and used by the community was remarkable: here was
a magic switch that allowed a researcher to turn down the
level of any gene of interest, specifically, and in vivo. Not
only was it a technology perfectly married to the genome
sequence, but it was also astoundingly easy and cheap. The
discovery that dsRNAs could be delivered toworms not only
by injection or soaking following in vitro synthesis, but that
feeding dsRNA-expressing bacteria to worms could
generate similar knockdown effects
[70
e
72]
paved the way
for the development of a genome-scale RNAi library of
dsRNA-expressing bacteria covering the great majority of
predicted genes
[66]
. With this library, simply by feeding
bacteria to worms, any researcher can target a specific gene
in one worm or in a billion, examine all the members of
a molecular pathway, target pairs of genes, or
e
Reverse Genetics and the Magic of RNAi
The worm has ~20 000 predicted coding genes. For each
gene, reverse genetics asks 'If I change the activity of this
gene, what is the effect on the organism?'. The key for
systems biology is to ask this comprehensively
most rele-
e
to
systematically perturb the activity of each and every pre-
dicted gene and examine the phenotypic effect. In S. cer-
evisiae and S. pombe, researchers have constructed
collections of strains in which each predicted gene has been
perturbed in a similar manner. For example, there are
deletion collections, in which each predicted gene has been
deleted by chromosome engineering
[58,59]
, and over-
expression collections in which each predicted open
reading frame is placed under promoter giving high
expression levels
[60,61]
. With such collections, rather than
use random mutagenesis to attempt to identify the genes
required for a specific process, one can scan the genome
a gene at a time and ask whether perturbing each and every
predicted gene individually affects the process of interest.
In this section, we examine the progress made in the worm
to establish analogous collections, and how the data
generated from such systematic surveys of gene function
have given key insights into the organization of biological
processes and the evolution of gene function.
vantly for system biology
carry out genome-scale RNAi
e
e
screens.
RNAi has completely transformed worm genetics, and
to date over 50 genome-scale screens have been carried out.
Each screen walks through the genome, a gene at a time,
and asks 'If I knock down expression of this gene by RNAi,
does it affect my process of interest?'. An RNAi screen,
then, is just like a classical genetics screen but without the
need for positional cloning and without random mutagen-
esis and the resulting uncertainty concerning coverage
e
every gene can be targeted, (almost) every gene can be
tested. Screens have been done for genes that affect crude
phenotypes such as viability or brood size
[66]
; for genes
that are involved in the development of specific tissues such
as the vulva (e.g.,
[73]
) or the neuromuscular system
[74]
;
and for genes that affect individual molecular processes
such as DNA damage response
[68]
, fat metabolism
[65]
,or
even RNAi itself
[75]
. The initial output of any screen is
a list of validated hits: this set of genes gives this specific
phenotype when knocked down by RNAi. These hits are
