Essential Cell Biology 5th edition

Genetics as an Experimental Tool
677
each well contains
E. coli expressing
a different dsRNA
wild type
(fertile)
C. elegans
ADD TO WELLS
WORMS INGEST E. coli;
RESULTING PHENOTYPES ARE
RECORDED AND ANALYZED
sterile
96-well plate
Figure 19−33 RNA interference provides
a convenient method for conducting
genome-wide genetic screens. In this
experiment, each well in this 96-well plate
is filled with E. coli that produce a different
double-stranded (ds), interfering RNA.
E. coli are a standard diet for C. elegans
raised in the laboratory. Each interfering
RNA matches the nucleotide sequence of
a single C. elegans gene. About 10 worms
are added to each well, where they ingest
the genetically modified bacteria. The plate
is incubated for several days, which gives
the RNAs time to bind to and inactivate
their target genes—and the worms time
to grow, mate, and produce offspring. The
plate is then examined in a microscope,
which can be controlled robotically, to
screen for genes that affect the worms’
ability to survive, reproduce, develop, and
behave. Because the investigator knows
which interfering RNA was added to each
well, the gene responsible for any resulting
defect can be readily identified. Shown here
are wild-type worms alongside a mutant
that shows an impaired ability to reproduce.
(Adapted from Lehner et al., Nat. Genet.
38:896–903, 2006.)
If the organism is diploid—a mouse or a pea plant, say—and the mutant
ECB5 e19.33/19.33
phenotype is recessive, there is a simple solution. Individuals that are
heterozygous for the mutation will have a normal phenotype and can be
propagated. When they are mated with one another, 25% of the progeny
will be homozygous mutants and will show the lethal mutant phenotype;
50% will be heterozygous carriers of the mutation like their parents and
can be used to maintain the breeding stock.
But what if the organism is haploid, as is the case for many yeast and
bacteria? One way to study lethal mutations in such organisms makes
use of conditional mutants, in which the protein product of the mutant
gene is only defective under certain conditions. For example, in mutants
that are temperature-sensitive, the protein functions normally within a
certain range of temperatures (called the permissive temperature) but
can be inactivated by a shift to a nonpermissive temperature outside this
range. Thus the abnormal phenotype can be switched on and off simply
by changing the temperature. A cell containing a temperature-sensitive
mutation in an essential gene can be propagated at the permissive temperature
and then be driven to display its mutant phenotype by a shift to
a nonpermissive temperature (Figure 19−34).
Many temperature-sensitive bacterial mutants were isolated to identify
the genes that encode the bacterial proteins required for DNA replication;
investigators treated large populations of bacteria with mutagens and
23ºC
mutagenized cells plated
out in Petri dish grow into
colonies at 23ºC
colonies replicated
onto two identical
plates and incubated
at two different
temperatures
23ºC
36ºC
mutant colony in which
cells proliferate at the cooler,
permissive temperature but
fail to proliferate at the warmer,
nonpermissive temperature
Figure 19−34 Temperature-sensitive
mutants are valuable for identifying
the genes and proteins involved
in essential cell processes. In this
example, yeast cells are treated with
a mutagen, spread on a culture plate
at a relatively cool temperature,
and allowed to proliferate to form
colonies. The colonies are then
transferred to two identical Petri
plates using a technique called
replica plating. One of these plates is
incubated at a cool temperature, the
other at a warmer temperature. Those
cells that contain a temperaturesensitive
mutation in a gene essential
for proliferation can be readily
identified, because they form a
colony only at the cooler, permissive
temperature.