Biomedical Engineering Reference
In-Depth Information
binding protein (CREB) and its binding site (cAMP response element, CRE) have
been suggested to play a major role in reporter gene assay. CREB belongs to the
family of basic leucine zipper transcription factors and modulates gene transcription
by binding to the CRE in the promoter regions of various genes. In the CRE-based
odorant sensing system, odorant binding to OR stimulates adenylyl cyclase (AC)
via G protein activation. Activated AC causes a rise in intracellular cAMP from
ATP, which activates protein kinase A (PKA). The catalytic unit of PKA can then
move into the nucleus and phosphorylate a CREB to CREB-P, allowing it to bind
to a CRE in the promoter of a target gene, thus increasing transcription [
63
,
67
].
Generally CRE/CREB-driven gene expression via ligand stimulation can be mea-
sured as luciferase activity or fluorescence protein expression. As mentioned above,
cAMP is the second messenger involved in olfactory signal transduction. Thus, it
would be of interest to measure cAMP since it is a molecule that transmits the signal
from the membrane to the metabolic machinery of the cytosol.
Katada and co-workers reported that odorant-induced cAMP response can be
observed using a CRE-regulated luciferase reporter gene assay [
52
]. A luciferase
reporter gene assay using the zif268 promoter, known to contain CREs, was adopted
to amplify the cAMP signals. An intracellular cAMP increase upon exposure to the
proper agonist initiates a cascade that leads to CRE-mediated gene expression. In
addition, odorant-induced cAMP increases via Gαs and AC activation can be mea-
sured directly using an enzyme-linked immunoassay [
43
]. Bioluminescence mea-
surements through a luciferase reporter were applied in response to odorant stimu-
lation [
68
]. Yeast functional activity, which co-expresses rat OR (rOR) I7 and G
olf
,
was evaluated by a luciferase reporter placed under an inducible promoter, triggered
by the specific odorant to its related OR interaction, and having the dose-response
curves, from 10
−12
to 10
−4
M of a specific odorant, plotted as a difference to negative
controls. The luciferase reporter can be used as a useful tool for simple, rapid odorant
screening of ORs to provide olfactory coding mechanisms. Fukutani and co-workers
reported a bioluminescence reporter-based 2,4-dinitrotoluene (DNT)-sensing system
using OR-expressing yeast [
69
]. A biomimetic odor-sensing system was constructed
with engineered yeast expressing OR, using firefly luciferase (luc) as the reporter.
In addition, Radhika et al. used
S. cerevisiae
to screen for the rat OR226 gene that
possesses sensitivity for DNT, an analog of the explosive trinitrotoluene [
66
] (see
Fig.
11.4
). This screening was accomplished by constructing a chimeric OR receptor
that both the N- and C-terminal regions of the endogenous protein replaced with the
rORI7 receptor. This system can be expected to enable the quantification of flavors,
fragrances, and/or the avoidance of harmful agents or explosive accidents.
11.2.3
Fluorescence and Bioluminescence Resonant Energy
Transfer-based Odorant Sensing
Recent progress in molecular biology has made several biotechnological tools such
as FRET and BRET available. These developments provide inroads to continuous
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