Advances in transgenic technology - Principles of Gene Manipulation - page 266

phenotypes, because the remaining wild-type copy
of the gene provides enough gene product for nor-
mal or near-normal activity. However, some loss-of-
function mutations are fully dominant over the
wild-type allele, because the mutant gene product
interferes with the activity of the wild-type protein.
Such mutants are known as dominant negatives , and
principally affect proteins that form dimers or larger
multimeric complexes.
The deliberate overexpression of dominant-
negative transgenes can be used to swamp a cell
with mutant forms of a particular protein, causing
all functional molecules to be mopped up into
inactive complexes. The microinjection of DNA con-
structs or in vitro -synthesized dominant-negative
RNA into Xenopus embryos has been widely used to
examine the functions of cell-surface receptors in
development, since many of these are dimeric (e.g.
see Amaya et al. 1991, Hemmati-Brivanlou &
Melton 1992). Dominant-negative proteins stably
expressed in mammalian cells have been used pre-
dominantly to study the control of cell growth and
proliferation. A dominant-negative ethylene receptor
from Arabidopsis has been shown to confer ethylene
insensitivity in transgenic tomato and petunia. The
effects of transgene expression included delayed fruit
ripening and flower senescence (Wilkinson et al.
1997).

genome (the transcriptome ) and all the proteins that
are made (the proteome ).
In other parts of the topic, we have already dis-
cussed a number of functional genomics techniques,
including microarrays (p. 116) and variants of the
yeast two-hybrid system (p. 169). However, perhaps
the most important way to establish a gene's func-
tion is to see what happens when that gene is either
mutated or inappropriately expressed in the context
of the whole organism. This chapter has focused
on the development of novel transgenic strategies
for the analysis of gene function in animals and
plants, but such approaches are only applicable to
individual genes. For functional genomics, tech-
nologies must be available that allow the high-
throughput analysis of gene function, which is the
only way we can begin to understand what the
10 000 - 40 000 genes in the genomes of higher
eukaryotic cells are for. Such technologies are
discussed below.

Insertional mutagenesis

Traditional techniques for generating mutations
involve the use of radiation or chemical mutagens.
These tend to generate point mutations, and isolat-
ing the genes corresponding to a particular mutant
phenotype can be a laborious task, particularly in
the large genomes of vertebrates and plants.
An alternative is to use DNA as a mutagen. The
genomes of most animals and plants contain trans-
posable elements - DNA elements that have the ability
to jump from site to site in the genome, occasionally
interrupting genes and causing mutations. Some of
the gene-silencing methods discussed in the previ-
ous section appear to be based on defence strategies,
many involving DNA methylation, that exist to resist
the movement of such elements in the genome. How-
ever, if these endogenous transposable elements can
be mobilized at a sufficient frequency, they can be
used to deliberately interrupt functional genes and
generate insertional mutants. Importantly, popula-
tions of animals or plants carrying transposable
elements can be set up for saturation mutagenesis,
such that a suitable number of transposition events
is induced to theoretically interrupt each gene in the
genome at least once somewhere in the population
(e.g. see Walbot 2000).

Transgenic technology for
functional genomics

In the last 5 years, the complete sequences of the
genomes of several important model eukaryotic
species have been published: the yeast S. cerevisiae
(Mewes et al. 1997), the nematode worm C. elegans
( C. elegans Sequencing Consortium 1998), the fruit
fly Drosophila melanogaster (Adams et al. 2000), the
model plant Arabidopsis thaliana ( Arabidopsis Genome
Initiative 2000) and, most recently, the human
genome itself (International Human Genome
Sequencing Consortium 2001, Venter et al. 2001).
With the wealth of information that has been gener-
ated by these genome projects, the next important
step is to find out what all the newly discovered
genes actually do. This is the burgeoning field of
functional genomics , which aims to determine the
function of all the transcribed sequences in the