Biology Reference
In-Depth Information
unsuited to establishing a dynamic model of differential gene expression at high tem-
poral resolution. Promoter fusion technology, however, allows dynamic changes in
gene expression to be established at high temporal resolution with relative ease and at
low cost. This approach involves establishing promoter activity by monitoring
expression of the
gfp
and
lux
reporter genes. Green fluorescent protein (GFP) from
Aequorea victoria
and the bioluminescent firefly and bacterial luciferase (Lux)
systems have revolutionized such expression studies. Cellular levels of GFP and
Lux can be established without perturbing the system under study. The activity of
promoters can be established at high temporal resolution because of the cellular
half-lives of GFP (very long) and Lux (very short). Moreover, promoter fusions
can be generated in an automated high-throughput manner, allowing gene expression
profiles to be established on a global scale.
In this chapter, we summarize the essential features of theGFP andLux reporter pro-
teins.We outline how they have been applied to establishingglobal changes in promoter
activity at high temporal resolution in
Escherichia coli
and
Bacillus subtilis
.Wethen
describe a methodology we have developed to generate promoter fusions in a high-
throughput manner and establish gene expression on a global scale with high reproduc-
ibility in
B. subtilis
that can easily be adapted for expression studies in other bacteria.
2
GENE FUSION TECHNOLOGY
2.1
History
The generation and use of reporter gene fusions has a long history in molecular genet-
ics (for review, see
Silhavy and Beckwith, 1985
). Some of the first fusions were gen-
erated by Benzer in his studies on the independently transcribed
r
IIA and B genes of
the T4 phage. A deletion extending from the carboxy end of the A gene to the amino
end of the B gene removed all expression signals of the B gene and generated a trans-
lational fusion where the hybrid
r
IIA-B protein retained some B activity (
Champe and
Benzer, 1962
). Mutations of the A gene could now be studied by measuring their effect
on B protein activity (
Silhavy and Beckwith, 1985
). This established the fundamental
principle of gene fusion technology, whereby the expression signals of any gene can be
studied by fusing them to a reporter gene whose product is easily assayable. Fusion
technology was further developed by combining knowledge of the
lac
operon with that
of mobile genetic elements such as phages and transposons. LacZ emerged as a
reporter of choice because
-galactosidase activity could easily be assayed using sub-
strates that yielded coloured compounds (
Slauch and Silhavy, 1991
). Thus a promoter-
less
lacZ
reporter gene was combined with modified Mu and
b
l
phages [e.g. Mu d1(
Ap
lac
)and
plac
Mu] that allowed the random generation of transcriptional fusions with
chromosomal promoters (for review, see
Silhavy and Beckwith, 1985
). The advent of
recombinant DNA technology brought precision and sophistication to the construction
of fusion generating vectors. Plasmids and transposons replaced the more cumbersome
phages as the genetic element of choice for the construction and mobilization of
fusions. Transcriptional and translational fusions could be rapidly generated with pre-
cision using the polymerase chain reaction (PCR). Fusion technology could be
l
