LUCIFERASE GENE AND ITS APPLICATIONS

Background
Bioluminescence is a form of light produced by a chemical reaction in living
organisms. The function of bioluminescence may vary from one organism to the other,
such as for defense against predators, for predation or for communication with their
mates . It is exhibited by a diverse group of organisms
although their number is very less compared to the total number of known species. It has
been estimated that luminous organisms may have come from about 30 different
evolutionarily distinct origins .
The enzyme luciferase and its substrate luciferin are discovered to be the agents
responsible for Bioluminescence. The biochemical and physiological mechanisms
responsible for the bioluminescence are different among the luminous organisms
. Hastings and Morin in 1991 have reported that the genes and
proteins responsible for bioluminescence are not evolutionarily conserved. These genes
and proteins are evolved independently and are not similar to each other.
Shimomura et al., first discovered luciferase in 1962. They extracted the light
emitting protein from jellyfish “Aequorea aequorea” and found the compound to be
emitting blue color instead of expected green color. This protein was later named as
“aequorin”. The expected green color was emitted by another protein, which absorbed the
blue color emitted by aequorin and re-emitted the energy at a higher wavelength. This
protein was later named “green fluorescence protein” (GFP).
There have been many studies on luciferase enzyme systems in different groups
of luminescent organisms. There are also many applications of this enzyme and the
associated GFP in molecular biology fields. In the following review the different aspects
of the enzyme luciferase and its applications will be addressed.
Chemistry
The chemistry behind bioluminescence has two major components. One is the
substrate which is generically called luciferin and the other is the enzyme that catalyses
the substrate to emit a photon called as luciferase.

The basic reaction involves luciferase catalyzing the oxidation of luciferin
resulting in light and an inactive "oxyluciferin" In most cases, fresh luciferin must be
brought into the system, either through the diet or by internal synthesis.
Luciferin + O2 Oxyluciferin + light
Luciferase
Sometimes the luciferin and luciferase (as well as a co-factor such as oxygen) are
bound together in a single unit called a "photoprotein". This molecule can be triggered to
produce light when a particular type of ion is added to the system (frequently
calcium).

Types of Luciferin
Luminous organisms are intensively studied for their difference in the luminous
systems. The luminous systems differ in the structures of the luciferins and luciferases,
although they have some properties in common. All known luciferases are oxygenases
and they oxidize the luciferin using molecular oxygen to intermediate enzyme bound
peroxide, which dissociates to produce an intermediate or product in its excited state to
emit photon. Luminous systems of luminous organisms are classified into four types
based on the different type of luciferin they possess. They are 1) Coelenterate luciferin
(Coelenterazine), 2) Bacterial luciferin, 3) Dinoflagellate luciferin, and 4) Firefly
luciferin.
Coelenterazine is the most popular of the marine luciferins, found in a variety of
phyla. This luciferin is biochemically similar to other cnidarian luminescent systems. It
possesses an imidazopyrazine skeleton and is widely distributed among the cnidarian
group.
Coelenterazine
Bacterial luciferin is a reduced riboflavin phosphate, which is oxidized in
association with a long-chain aldehyde, oxygen, and a luciferase. Luminous bacteria are
observed ubiquitously in seawater samples. All bacterial luciferases are heterodimeric
containing two polypeptides alpha and beta. Fisher et al. in 1995 reported the X-ray
structure of luciferases.
Bacterial Luciferin

Dinoflagellate luciferin is a novel tetrapyrrole related to chlorophyll. In the genus
Gonyaulax, at pH 8 the molecule is "protected" from the luciferase by a "luciferinbinding
protein", but when the pH lowers to around 6, the free luciferin reacts and light is
produced. A modified form of this luciferin is also found in herbivorous euphausiid
shrimp, perhaps indicating a dietary link for the acquisition of luciferin.

Firefly luciferin is a unique benzothiazolethat requires ATP to form luciferyl
adenylate intermediate. The adenylate then reacts with with oxygen to form a cyclic
luciferyl perooxy speces, which breaks down to yield CO2 and an excited state of the
carbonyl product. Wood in 1995 reported that the long chain acyl-CoA synthase is
homologous with the firefly luciferase indicating the evolutionary origin of the gene

Luciferase Gene organisation
Bacterial luciferase gene
Luciferase genes are cloned and sequenced from luminescent bacteria of Vibrio,
Xenorhabdus and Photobacterium genera (Szittner, 1990). Two genes code for the two á
and â subunits of bacterial luciferase , named luxA and luxB respectively. The
luciferase genes occur as two major operons, also containing other proteins associated
with the luciferase activity that is involved in the synthesis of myristic aldehyde
(Meighen, 1991) . The operon also contains genes that control the development
and expression of luminescence by a mechanism of “autoinduction” (Nealson et al.,
1970). The luciferase operon contains about 10 genes and they are transcribed only by the
presence of an autoinducer which is a homoserine lactone produced by the gene present
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in the operon itself named luxI gene. The ecological importance of this mechanism was
discovered in 1994 by Fuqua et al., who dubbed this phenomenon to be Quarum sensing.
It is also discovered that the autoinducer is responsible for the cell-cell signaling in
luminous bacteria.
Plate 2 Bacterial luciferase

Dinoflagellate luciferase gene
The Gonyaulax polyedra luciferin binding protein (LBP) gene and the luciferase
gene are the first dinoflagellate luciferase gene cloned and sequenced by Lee et al.,
(1993) and Bae and Hastings (1994) respectively. They sequenced the cDNA of the LBP
gene and found that the open reading frame of the gene is 2004 nucleotides in length and
has no introns. It is also found that it has no polyadenylation sites. The open reading
frame codes for about 688 aminoacids (~ 75kDa). The interesting circadian phenomenon
is seen in the dinoflagellate LBP gene, which causes the gene to be transcribed only once
a day and the protein destroyed every day (Hastings et al., 1993).
The luciferase gene was found to be 4.1kb and the luciferase enzyme is 130kDa.
This gene also did not have any introns. Cloning of the cDNA of the gene in Escherichia
coli showed the expression of the active luciferase protein (Bae and Hastings, 1994). It
was found that the LBP is responsible for the pH dependent binding of luciferin and the
luciferase is responsible for the oxidation of the luciferin to produce light. Li et al.,
(1997) reported a unique case of a 137kDa protein gene responsible of catalyzing the
light-emitting oxidation of the dinoflagellate luciferin. They also found that the gene had
three tandem repeat sequences of (~377, 377 and 379 bp) with no intervals, capable of
producing individually active luciferase.

Okamoto et al., in 2001 reported three new classes of the luciferase gene in new
members of dinoflagellate, Pyrocystis lunula and Lingulodinium polyedrum. The three
genes are named lcfA, lcfB and lcfC, which are unique and not related to the luciferase
genes of other dinoflagellates. The lcfA and lcfB genes are connected by a 2.2kb
intergenic region. In P.lunula, the lcfC gene has an intron (Figure 5). They also found the
presence of an intron in lcfC of P. lunula luciferase genes; different sizes of untranslated
regions in P. lunula and L. polyedrum luciferase genes, sharing no significant sequence
similarities; and notable differences in the frequency of synonymous substitutions in the
center of the repeat luciferase domains between the two species

Firefly luciferase gene
The Firefly luciferase gene was first cloned and sequenced by Wet et al., in 1985
from Photuris pennsylvanica, belonging to the Photurinae subfamily of the Lampyridae.
The gene sequence was about 1.8kb in length, coding for 545 aminoacid . The 5'
noncoding region was 61 bp and the 3' noncoding region is 135 bp in length. The
poly(A)tail was 24-nucleotide in length (Figure 6). Wood in 1995 studied the homology
of the luciferase enzyme aminoacid sequences with other known enzyme sequences. He
showed the enzyme’s similarity with adenylate kinase, the putative AMP-binding
domain, luciferin 4-monooxygenase, 4-coumarate CoA ligase, long-chain fatty acid CoA
ligase, 2-acylglycerophosphoethanolamine acyltransferase, the microbody-directing
sequence, peptide-synthesizing complexes, and acyladenylate-synthesizing enzymes.
Choi et al., in 2003 studied the luciferase gene organization in the Hotaria-group of
fireflies. They also studied the phylogenetic diversity of the luciferase gene in fireflies of
the Hotaria group.
Plate

Applications of Luciferase
Initial applications of luciferase were dependent on the type substrate that were
used for emitting light. The apoaequorin, which functions like the luciferase in
coelenterate system, was used to monitor intracellular calcium levels (Tanahashi et al.,
1990). Saran et al., (1994) used cnidarian luciferases to monitor intracellular calcium
changes in response to cyclic adenosinemonophosphate (cAMP) stimulation. Johnson et
al., (1995) used coelenterate luciferased to investigate the circadian changes in free
cytosolic calcium level in the chloroplasts of tobacco and Arabidopsis. Similarly bacterial
lux genes have been used as reporters for visualizing gene expression in Streptomyces
(Schauer et al., 1988) and to study the circadian transcriptional regulation in
cyanobacteria (Kondo et al., 1994). The firefly luciferase was widely used to measure
ATP (Hastings et al., 1997). Recently a combined use of two different luminous systems,
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such as firefly and Renilla luciferase enzymes are used to assay two different luciferins
has been reported by Sherf et al., 1997.
Luciferases are nowadays successfully used as reporter genes for gene expression
studies in living cells. Their application is growing in all aspects of the biological field
such as Animal research, environmental and plant research.
Luciferase in Animal Research
Molecular imaging of live cells
Luciferase is widely used in molecular imaging of cells in vivo. Luciferases are
used to track the in vivo behaviour of any tissue. Bhaumik and Gambhir (2002) validated
that the bioluminescence from Renilla luciferase (rluc) and the firefly luciferase enzyme
both can be imaged using different substrates for each and their kinetics can be studied in
vivo in the tissue. They carried out the experiment in mice by transfecting the mice with
C6 cells containing Firefly luciferase (fluc) and (rluc) (Plate 4).
Plate 4 Kinetics of light production from mice carrying s.c. C6-Fluc and C6-Rluc cells after simultaneous tail-vein injection of both Dluciferin
and coelenterazine. A mouse was injected s.c. with C6-Fluc (A), C6-Rluc (B), and C6 control cells (C) on right forearm, left
forearm, and right thigh regions, respectively. Simultaneous injection of both coelenterazine and D-luciferin mixture via tail-vein
shows bioluminescence from both the sites simultaneously but with distinct kinetics. A series of image at 2-min intervals is shown
from the same mouse. Each image represents a scan time of 1 min. The signal from C6-Rluc cells (B) peaks early and is near
extinguished within 10 min. Bioluminescence from C6-Fluc cells (A) shows a relatively strong signal beyond 10 min. The region of
control cells does not show any significant bioluminescence. R and L represent the right and left side of the mouse resting in supine
position.
Applications in Gene therapies
Molecular imaging of reporter therapeutic genes could play a critical role in gene
therapy to achieve controlled and effective delivery of genes to target cells avoiding
ectopic expression. Luciferases play a major role in reporting the level, the location and
the duration of the gene expression. A study by Weng et al., (2000) used somatic cell
gene transfer of the fluc linked heme oxygenase-1 (HO-1) transgene to the lung alveoli of
neonatal mice through transpulmonary injection. They monitored the gene expression in
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real time and were able to detect targeted expression of the gene in the cells of the
alveolar epithelium rather than other cells of the lung

Application in transgenic animal research
A study carried out by Voojis et al., (2002) involved the use of a conditional
mouse model for retinoblastoma suppressor gene-dependent pituitary cancer development
with co-expression of fluc genes enabling long term bioluminescence imaging,
quantification of tumour and assessment of chemotherapeutic response(Plate 6) .
Plate 6 Pseudocolor images of longitudinal tumor growth in a POMCcre-POMCluc;RbF19/F19 animal.
Quantification of theemitted photons shows an approximately exponential tumor growth between 12 and 16 weeks.
Another study carried out by Carlsen et al., (2002) developed transgenic mice that
express the fluc gene under the control of the nuclear factor 6B (NF-6B) promoter. This
enabled then for real-time imaging of the NF-6B promoter activity and its modulation in
living mice (Plate 7

Luciferases in Environmental research
Applications as biosensors
Luciferase reporter systems are developed into luminescent bacterial biosensors to
detect a wide range of pollutants and also to assess the bioavailability in the
environmental samples. Hollis et al., in 1999 transformed a Saccharomyces cerevisiae
with firefly luciferase gene from Photinus pyralis to be used as a biosensor. They studied
the cell health upon exposure to toxins directly correlating with the endogenous supply of
energy by ATP. A bioluminescent plasmid reporter that shows a sensitive response
towards 2-4-Dichlorophenoxyacetic acid and 2, 4-Dichlorophenol in soil was constructed
and introduced into the chromosome of Ralstonia eutropha. The transformed bacterium is
capable of detecting the concentrations of 1.2mM 2,4-D and 1.1X102 μM of 2,4-
dichlorophenol (Hay et al., 2000). Pellinen et al., (2002) developed Escherichia coli K-12
strain for specific detection of the tetracycline family of antimicrobial agents. It was
optimized to work with fish samples. The biosensing strain contains a plasmid
incorporating the bacterial luciferase operon of Photorhabdus luminescens under the
control of the tetracycline responsive element from transposon Tn10. The lowest levels of
detection of tetracycline and oxytetracycline from spiked fish tissue were 20 and 50
μg/kg, respectively, in a 2-h assay.