Table of Contents

clattergumneckBiotechnology

Oct 23, 2013 (3 years and 5 months ago)

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Original
Author: R
Appanah




1



Table of Contents

1.

Introduction


2

2. Examples of Reporter Genes


3

2.1 Chloramphenicol acetyltransferase (CAT)

3

2.2 β
-
galactosidase

4

2.3 β
-
lactamase

5

2.4 Alkaline Phosphatase
s

7

2.5 Luciferase
s

7

2.6 Green Fluorescent Protein (GFP)

8

2.7 Aequorin

9

2.8 Yeast
two
-
hybrid System

9

3. Applications of Reporter Gene Systems


10

3.1 Monitoring the Efficiency of Gene Transfer

10

3.2
Characterizing Protein/Protein and
Ligand/Receptor interactions

11

3.3
Screening for Discovery of Novel Drugs and for Toxicological Studies

11

4.
Comparing the usefulness of the Yeast
-
two
-
hybrid system and that of co
-
immunoprecipitation to elucidate protein
-
protein interactions.


11

5.
Critical Appraisal of Research Articles using Reporter Gene Technology.


13

5.1 Elaboration of a monocistronic dual reporter to evaluate the replication of hepatitis
C virus.

13

5.2
Using luciferase reporter gene assays to characterize the gene expression of
the
two
mini open reading frames (uORFs) found in the obscure p13 gene in

the

Helicoverpa armigera

nucleopolyhedrovirus
.

17

6. References


22


Preview:

To fulfil the requirements for the ‘Techniques Sessions’ Module (03 18593), students need to (i)
provide details of the scientific basis of a technique including
the
advantages and disadvantages in
comparison with a similar technique and (ii) to provide a
critical appraisal of two research papers
that include the topic of interest as a central part of the research, including how the technique has
enhanced t
he findings within each paper.
Herein, a comprehensive analysis of reporter gene
technology is present
ed.
Sections 1
-
3 introduce the concept of reporter genes and describe different
reporter genes, their relative usefulness and limitations. The broad spectrum of applications of
reporter gene technology is also illustrated. In section 4, we depict how the y
east
-
two hybrid
reporter assay compares to co
-
immun
o
precipitation for the study of protein
-
protein intera
ctions.
Fin
ally,

in section 5,

two studies (based largely
on

reporter gene technology)

are critically reviewed.
The first article by Wu
et al.

(2011) details the production of a dual reporter gene to monitor
hepa
titis C infections in host cell cultures
.
The second publication describes how luciferase reporter
gene assays were used to characterize the gene expression of mini open reading frames
f
ound in the
obscure p13 gene of

the

Helicoverpa armigera

nucleopolyhedrovirus (Qiao
et al.

2011).


Original
Author: R
Appanah




2



1.

Introduction
.

Alterations in gene expression patterns can
mediate or regulate a number of cellular phenomena
including, but not restricted to, cellular sign
alling, development and proliferation. Reporter gene
technology is concerned with identifying (and ideally, quantifying) desired alterations in gene
expressio
n, primarily to study the roles

of
specific genes

and
to characterize
protein
-
protein
interactions

(
Ji
a
n
g
et al.

2008).

To achieve this goal
,
the reporter
-

an easily identifiable marker
-

is
linked or attached to a regulatory sequence

of interest
.
Thus, the gene or its gene products
(depending on the nature of the reporter) will be tagged
with the repo
rter
which renders them
identifiable

(Fig

1)
.
Usually, the characteristics of the reporter gene are visually
demarking (Naylor,
1999). Other sought characteristics of the reporter are high sensitivity and dynamic range,
low
cost,
reliability and
convenience of use

(for example, the readiness and ease of detection of the reporter)
.

In principle, however, the overriding factor when settling on a particular reporter is its ‘foreignness’
from the biological sample to be tested.

Indeed, if the reporter

is

fully
exogeno
us (non
-
self) to the
biological system to which it is applied,
the possibility of background activity and, consequently, of
false positives

will inherently be reduced

(Jiang
et al.

2008).
Furthermore, the choice of reporter is
dependent on

the very nature of the empirical set
-
up. For instance, experiments dealing with gene
expression will require different reporters compared to experiments

concerned with the monitoring
of transfection
-
efficiencies.




Fi gure 1: A si mpl i sti c fl ow
-
chart

hi ghl
ighti ng the
general
worki ng pri nci pl e behi nd

the reporter gene
technol ogy. Image constructed usi ng Powerpoi nt.

Since the introduction of the first reporter gene (
β
-
galactosidase), a number of other reporter genes
have been elaborated, extending the
r
ange of
possible applications of

the technology. In this review,
we describe some of the more popular reporter genes
, discuss their relative advantages and
disadvantages

and

depict the spectrum of applications of the technology in the field of basic biology
and advanced biotechnology.
Two

recent scientific studies (which made extensive use of

reporter
gene technology
) are also reviewed. T
he background
, scientific accomplishme
nt

and impact

of these
two studies are discuss
ed to some extent
.



Original
Author: R
Appanah




3



2.

Examples of Reporter Genes
.

Herein is a list of some

of the best
-
characterized and most pop
ular reporter gene systems. As well as
summarizing the advantages and drawbacks of each individu
al technique, we attempt to

succinctly
describe some of their

recent
developments
.
However, t
he scope of this paper is limited but
excellent reviews on reporter gene systems are otherwise available (Jiang, 2008; Naylor, 1999; New
et al.
2003).
Table 1
give
s a brief overview of several reporter genes and their characteristics.

Tabl e 1.
Reporter genes and thei r scored characteri sti cs.
+++ (hi ghl y appropri ate), ++ (appropri ate), + (ti me
-
consumi ng) or l abo
u
r
-
i ntensive, (

) i nappropriate. Qureshi (2007).



2.1
Chloramphenicol acetyltransferase (CAT)

Chloramphenicol
O
-
acetyltransferase
(CAT)
is an enzyme found exclusively in prokaryotes. It
catalyzes
the transf
er of
(one or two)
acetyl groups from acetyl
-
CoA
molecules
to chloramphenicol,
detoxifying the latte
r in the process.

The CAT gene product is stable and is detectable at attomolar
(10
-
18
) levels

(New
et al.

2003)
.

Thus, the CAT reporter gene is ideally suited for mammalian systems.
In fact, CAT was the first gene reporter system used to analyse
transcri
ption
-
regulation events

in
mammalian cells. There are several alternative detection protocols suitable for the CAT reporter
gene. In its earliest iteration, acetyl CoA and radioactively
-
labelled
14
C
-
chloramphenicol were
incubated with the transformed biol
ogical sample.
Separation of chloramphenic
ol and its acetyl
derivatives was

performed via thin layer chromatography (TLC) and autoradiography

(Fig 2.A)
.
Nordeen
et al.

(1987)
then
improved the protocol by using radioactively
-
labelled acetyl
-
CoA

instead
.
However, the use of radioisotopes limits the usefulness of CAT in living cells. As such, an automated
ELIS
A assay has been introduced but the

linear range and sensitivity
of the latter can be

deplored

(Bronstein
et al.

1994; Suto & Ignar, 1997)
. A CAT assa
y incorporating a fluorophore (BODIPY 1
-
deoxychloramphenicol) has also been developed

since

(Fig 2.B)

(Lefevre
et al.

1995)
. While the
sens
itivities

of the fluorescent and radioactive assays are comparable, chromatographic separation is
easier in the forme
r. CAT assays have been extensively used to investigate the patterns of altered
gene expression in disease models, including heart disease

(Selbert & Rannie,
2002)
. Because of the
dearth of simplistic separation and detection protocols, CAT assays cannot b
e applied to high
-
throughput studies

(New
et al.

2003)
.


Original
Author: R
Appanah




4







Fi gure 2. A:
After compl eti on of the CAT, the products are separated vi a TLC and exposed to X
-
rays. In the
exampl e provi ded, l ane 1 i s the promoter 1 construct, l ane 2 i s the promoter 2 construct, l ane 3 i s the negati ve
control and l ane 4
,

the posi ti ve control. The pre
sence of radi oacti ve acetyl ated chl orampheni col mol ecul es
corresponds to acti ve expressi on of
the
gene of i nterest. Image source: Davi dson Col l ege (2002). B: The
fl uorescent BODIPY 1
-
deoxychl orampheni col i s acetyl ated by CAT, l i ke chl orampheni col i tsel f. H
ere, the green
-
fl uorescent BODIPY FL 1
-
deoxychl orampheni col marketed by Invi trogen i s shown (Li fe Technol ogi es, 2012).

2.2
β
-
Galactosidase

The bacterial lacZ gene codes for β
-
galactosidase which has been ext
ensively used as a reporter
,
particular to assess

the efficiency of transfection in prokaryotes. Simple colorimetric assays using
substrates of β
-
galactosidase, such as ONPG (o
-
nitrophenyl
-
β
-
D
-
galactopyranoside) and X
-
Gal (5
-
bromo
-
4
-
chloro
-
3
-
indolyl galactoside)
, have been developed but these display poo
r sensitivities and
narrow dynamic ranges (Jiang
et al.

2008; Naylor, 1999)
. ONPG and X
-
Gal have since been
superseded by more sensitive
substrates with adaptable biological and/or chemical

properties, such
as
luminescence

(New
et al.

2003)
.

For example,
Wehrman
et al.

(2006) have designed a sequential
reporter
-
enzyme luminescence (SRL) protocol

for the detection of β
-
galactosidase activity
in vivo
.

β
-
galactosidase first cleaves a caged D
-
luciferin
-
galactoside conjugate (Lugal) freeing D
-
luciferin. The
la
tter
interacts with firefly luciferase to produce light (Fig 3.A).
Fig 3.B demonstrates the sensitivity
of the method. Elsewhere, β
-
galactosidase has been used as a contrast agent for MRI (magnetic
resonance imaging)
to monitor gene expression
(Louie
et a
l.

2000).

The downside of β
-
galactosidase
reporter gene
s is that the enzyme is endogen
ous
ly expressed in mammalian cells. Alkaline pHs do
specifically
inhibit the activity of

mammalian

(but not exogenous)
β
-
galactosidase but this inhibition
is often
incomplete, so that the activity of the exogenous enzyme may suffer from noise.

Acetyl CoA

A

B

Original
Author: R
Appanah




5






Fi gure 3. A:
The mol ecul e gal actosi de
-
luciferi n (
Lugal
)

i s not a
di rect substrate for fi refl y l
uc
i ferase, but when
cl eaved by β
-
gal actosi dase,
l uci ferin

i s freed from the conjugate. Thi s l uci feri n mol ecul e i nteracts enzymati cally
wi th fi refl y l uci ferase to produce photons. B:
FLuc
-
expressi ng cel l s were i njected i nto the l eft l eg of mi ce and
cel l s expressi ng both l acZ and l uci ferase were i njected i nto the
ri ght l eg. Introducti on of l ugal 6 h
ours

after the
i ni ti al i njecti on resul ted i n a pronounced l umi nescent response i n the ri ght l eg i njected but onl y a mi ni mal
l umi nescence si gnal was detected i n the l eft l eg. As a control, l uci feri n was i njected 24 hours
after the i ni ti al
i njecti on. The resul ti ng bi ol umi nescence

i n both l egs was

comparabl e, demonstrati ng the dependence of Fl uc
-
generated l umi nescence on the presence of β
-
gal actosidase. Images
obtai ned from

V
on Degenfel d
et al.

(2009) and

Wehrman
et al.

(2006).

2.3
β
-
lactamase


β
-
lactamases

are enzymes secreted by Gram negative bacteria that digest
β
-
lactam antibiotics such
as penicillin

and cepha
losporin

(New
et al.

2003)
.
β
-
lactamases

are
grouped

into 4 classes (A
-
D)
according to their structures, the
range of substrate
s

they act upon and their response to inhibitors
(Jiang
et al.

2008).
The
β
-
lacatamase reporter gene makes use of TEM
-
1
β
-
lacatmase (Bla
)

(a
member of the class A)
encoded

by

the ampicillin resistance gene from

Escherichia coli
.
There is no
endogenous
Bla

in mammalian cells nor is there any eukaryotic ortholog to the enzyme (Qureshi,
2007).
It

is not toxic to mammalian cells, even when over
-
expressed (Campbell, 2004; Qureshi,
2007).
The contemporary working principle of the

Bla

r
eporter gene depends on FRET (fluorescent
resonance energy transfer) and a membrane
-
permeable precursor molecule which only becomes

active


after being
entrapped in the

cytoplasm (Fig 4).
T
he substrate used
[
CCF2
]

is made up of two
distinct fluorescent dy
es: 7
-
hydroxycoumarin
-
3
-
carboxamide (henceforth referred to as coumarin)
and
fluorescein held together by a cephalosporin

(Zlokarnik
et al.

1998)
.
CCF4 is similar to CCF2 but
is more soluble and, hence, is preferred in some situations (Qureshi, 2007).
This

substrate is
esterified (with an acetoxymethyl group, AM). The acetoxymethylated
-
CCF2/4 can penetrate the
cellular membrane by passive diffusion but non
-
specific intracellular enzymes remove the
acetoxymethyl (ester) group. As a consequence, CCF2/4 assume
s a negative charge and can no
A

B

Original
Author: R
Appanah




6



longer escape the cytoplasm. Upon excitation at 409 nm, the donor dye, coumarin, transfer energy
to the

recept
or dye, fluorescein which relaxes and emits light at 518 nm

(
at
lower energy)
.

When Bla

is expressed from the transgene, however, it cleaves the cephalosporin segment of CCF2/4,
liberating fluorescein from coumarin. Hence, after the cleavage, excitation of coumarin does not
lead to energy transfer.
Instead, t
he coumarin relaxes and emits lig
ht at 447 nm.

Thus,
in
the
presence of Bla

(and of the transgene of interest)
the biological sample will stain blue instead of
green (Fig 4).
In fact, this change of colour can be monitored live so that cells bearing the

Bla
-
transgene can be identified sim
ply and categorically by using epifluorescence microscopy. Moreover,
unlike luciferases and aequorin (discussed below),

the Bla
-
gene reporter is compatible to flow
cytometery and
fluorescent activated cell
-
sorting (FACS) as the fluorescence is not transien
t

(Knapp
et al.

2003)
.
This reporter system is also very sensitive as the lower limit of detection is 50 Bla
molecules per cell (Zlokarnik
et al.

1998).

Bla
-
reporter gene technology is amenable to HTS (Hancock
et al.

2009) and, on top of
the

cytosolic form

discussed
, the BLA enzyme has been engineered into
secretable
-

(akin to SEAP/SPAP; discussed below) and membrane
-
associated variants. Consequently,
Bla

is an ideal reporter for eukaryotic gene expression (
Moore
et al.

1997;
Qureshi, 2007)
.




Intact CCF2 (Green colour at 518 nm
)



Cleaved CCF2 (Blue colour at 447 nm)

Fi gure 4:
C
CF2 i s the true substrate of Bl a
. An esteri fi ed vari ant of CCF2 (CCF2
-
AM, a precursor of sorts) i s abl e
to penetrate the cel l membrane (shown as a yel l ow l i ne) and cyt
opl asmi c esterases convert the CCF2
-
AM i nto
CCF2.
CCF2 i s negati vel y charged and, hence, cannot approach

the cel l membrane. It i s effecti vel y trapped
wi thi n

the cel l.
In i ntact CCF2, exci tati on of the coumari n dye l eads to an energy transfer by FRET to the

fl uoresc
e
i n dye whi ch ul ti matel y rel axes
,

l i berati ng photons correspondi ng to green l i ght (518 nm). If β
-
l acatamase from a transgene i s expressed
, however, the CCF2 i s di gested

l eadi ng to di ssociati on
of

the two
dyes. Thus, exci ta
ti on of coumari n at 409 n
m

does not l ead to FRET. Rather, the coumari n rel axes, produc
i ng
bl ue l i ght (447 nm)
.

CCF4 can be used i nstead of CCF2.
Image adapted from
Qureshi (2007).


Original
Author: R
Appanah




7



2.4 Alkaline phosphatase
s

Alkaline phosphatases are hydrolase enzymes responsible for the dephosphorylation of diverse
molecular entities including
nu
cleotides and proteins (Jiang
e
t al.

2008). They are so
-
named as they
display optimum activity at high (alkaline) pHs. Given their p
romiscuity, a number of reporter gene
assays have been developed from alkaline phosphatases.
Classicall
y, chromogenic

or fluorescent

substrates such as p
-
nitrophenyl phosphate (
PNPP) and flavin adenine dinucleotide phosphate
(FADP)
were used (Harbron
et al
.

1992; Schenborn

&

Groskreut
1999). Chemoluminescent
substrates have since been introduced (e.g. phenyl phosphate
-
substituted dioxethane, CSPD) and
the latters can be automated, making them particularly suited for studies involving high
-
throughput
screeni
ngs
(HTS)
(Naylor, 1999). A useful derivative of alkaline phosphatases is the secretable form

of alkaline phosphatase, SEAP (alternatively termed as secreted placental alkaline phosphatase,
SPAP). SEAP/SPAP is a mutant of the placental alkaline phosphatase

which lacks an anchorage
domain (New
et al.

2003) so that it is secreted in
to

the culture medium. Thus, pooling of culture
medium is sufficient to assess the
quantity of the
reporter gene with no need to disturb the growing
cells. This system has been used to monitor
inflammatory events and for HTS applications
(Hiramatsu
et al.

2006; Durocher
et al.

2000).


The major disadvantage of alkaline phosphatase as a
reporter gene is
that it is endogenously expressed in almost all organisms and cell
-
types, limiting its
application because of potential noise. However, two simple strategies are available to distinguish
between the activities of exogenous and

endogenous alkaline phosphata
ses. These are

thermal
selection and, alternatively, treatment of

cell samples with L
-
homoarginine. This is because
SEAP/SPAP is tolerant to both L
-
homoarginine and temperatures of up to 65

C, unlike endogenous
alkaline phosphatases (
Bronstein
et al.

1994)
.

2
.5 Luciferases

Luciferases are enzymes that oxidize various compounds, including luciferin and coelenterazine,
producing light in the process (Naylor, 1999).
The luminescence produced is proportional to the
enzymatic activity, providing a rapid diagnos
is of gene expression.
Hence, they can be used as
reporter genes.
Indeed, luciferase assays are capable of detecting as little as 10
-
20
moles of enzymes,

and are, consequently,

substantially more sensitive than chloramphenicol acetyltransferase reporter
as
says (Alam & Cook, 1990).
Bacterial luciferases oxid
ize

aliphatic aldehydes to the corresponding
carboxylates producing cyan light (Fig 5.A). These luciferases have a linear range of only 3 orders of
magnitude and are not resistant to thermal damage, limit
ing their usefulness in mammalian systems
(Naylor, 1999; New
et al.

2003)
. Fortunately
, f
irefly luciferase (cloned from
Photinus pyalis
)

has a
linear range of up to 8 orders of magnitude and is less susceptible to thermal damage

(Hawkin
s
et al.

1999).

This luciferase oxidizes D
-
luciferin releasing green light (560 nm). A limiting aspect of firefly
luciferase is that the light produced is transient
, but the protocol has been modified so as to
lengthen the duration of the flash and stabilize its signal i
ntensity (George
et al.

1997; George
et al.
1998). Thus, the signal can be detected through scintillation counters, enabling HTS applications.
However, the modified signal is still too transient for use in flow cytometry (Qureshi, 2007). A useful
alternati
ve to the firefly luciferase is the Renilla luciferase (cloned from
Renilla reniformis
) which
oxidizes coelentrazine (like aequorin; discussed below) to produce violet light (430
-
480 nm) (Fig 5.C).
This luciferase is particularly appropriate for

use in mam
malian systems as its

substrate,
coelenterazine, is naturally membrane
-
permeable so that cells need not be disturbed (Lorenz
et al.

1996).

Original
Author: R
Appanah




8





Fi gure 5. The mechani sms of acti on, the wavel ength
s

of l i ght emi tted and the substrates of (A) bacteri al
l uci ferase, (B) fi refl y l uci ferase and (C)
Renilla

l uci ferase. Image from Ji ang
et al.

(2008).



2.6 Green Fluorescent Protein

(GFP)

Green fluorescent proteins (GFPs) are known to exist in a number of
organisms (e.g.
Aequorea
,
Obelia
and
Renilla
) but, as of 2008, only the GFP gene from the jellyfish
Aequorea victoria

had

been
successfully cloned (Jiang
et al.

2008; Prasher
et al.

1992; Tsien, 1998).

The latter has been widely
used to visualize patterns
of gene expression
in vivo

(Fig 6
.A) and

to monitor protein localization and
transit (Soboleski
et al.

2005). The orbital electron of
GFP
is excited at 395 nm and, after it returns to
its ground state, it emits photons corresponding to green light at 509 n
m (Jinag
et al.

2008; New
et
al.

2003). The advantages of the GFP are numerous. Being inherently fluorescent, it does not require
the use of substrates

or co
-
factors
. I
t is non
-
invasive so

the cells do not need to be lyzed

and, hence,
it
is appropriate for

in vivo

use (Qureshi, 2007).
The one major disadvantage of GFP is that since the
process does not depend on enzymatic amplification, the limit of det
ection is only
about 10
5

molecules/cell

(Qures
h
i, 2007)
. This corresponds to a low sensitivity, limiting the usefulness of GFP.
However, through induced mutations and engineering, new derivative proteins have been produced
such as the enhanced GFP as well as yellow, far
-
red and even
cyan fluorescent proteins (
Fig 6.
B
-
D
).



A

B

C

Original
Author: R
Appanah




9








Fi g
ure

6
.A:
A young adul t Drosophi l a mel anogaster (approximatel y 6 hours ol d) expressi ng the bal ancer FKG34
gene tagged wi th GFP (Casso
et al.

1999).
B:

A d
ual
,

non
-
i nvasi ve reporter vi sualizati on i n mi ce

embryos
.
The
transgeni c ani mal s were

hemi z
ygous for ei ther the CK6/ECFP (ri ght)

or
YC5/EYFP

(l eft)

transgenes.

Thi s
doubl e
exposure
was obtai ned after consecuti vel y appl yi ng

ECFP and EYFP fi l ters

(Hadjantonakis
et al.

2002) ECFP
stands for enhanced cyan fl uorescent protei n and EYFP stands for enhanced yel l ow fl uorescent protei n.

C:
The
author

h
as

engi neered enhanced red and far
-
red fl uorescent protei ns

for
in vivo

i magi ng
. The i mage herei n
shows a HeLa cel l

expressi ng
fusi ons of TagRFP
(a red fl uorescent protei n) and
α
-
tubul i n

(Kel manson, 2009)
.
D:
Mul ti pl e cyan fl uorescent protei ns have been engi neered. The i mage here shows one such fl uorescent protei n
(mCerul ean
-
3) whi ch emi ts a cerul ean col our post
-
exci tati on. Here,
human
α
-
tubul i n
-
6 i s tagged wi th the
reporter gene (Markwardt
et al.

2011).

The purpose of thi s composi te i mage i s to depi ct the range of
appl i cations of fl uorescent protei ns as reporter genes, the spectrum of col ours avai lable and the appl i cability
of the

techni que to i ndi vi dual cel ls, as wel l as to embryos and to adul t organi sms.

2.
7 Aequorin

Like GFP, aequorin has been isolated from
Aequorea victoria
. It is a photoprotein made up of
apoaequorin (the apoprotein), coelenterazine (
the
prosthetic group) and oxygen (Inouye
et al.

1985).
In the presence of calcium ions [Ca
2+
]
, the conformation of the protein is altered in such a way
as to allow the oxidation of the coelenterazine. Subsequent de
-
excitation of the coelenterazine
liberates ph
otons at 469 nm (corresponding to a blue colour). The intensity of the signal is
dependent on the levels of Ca
2+

available for the reaction (New
et al.

2003). Like GFP, it is a non
-
invasive technique easily monitored thr
ough luminometry (Brini
et al.

1995)
. Additionally, its
sensitivity is comparable to chloramphenicol acetyltransferase
when used as a
reporter gene system

in mammalian cells

(Tanahashi
et al.

1990)

and it entails low levels of noise
.
However, unlike the
autofluorescent GFP, the presence of C
a
2+

and coelenterazine are necessary. The blue flash
produced from the oxidation process is also very transient (less than 5 seconds), requiring expensive
luminometers capable of detecting such rapid changes in luminosity (New
et al.

2003).


2.8 Yeast
two
-
hybrid System

The yeast two
-
hybrid (Y2H) system is a powerful tool used to identify and characterize protein
-
protein interactions (Hollingsworth & White, 2004; Stellberger
et al.

2010). The Y2H system involves
the expression of the protein of interest fuse
d to the DNA binding moiety of a transcription factor.
This molecule is classed as the ‘bait’. The second component of the Y2H system is either a single
target protein or a library of pro
teins fused to

the

activation

domain
of the same transcription
factor.

The
s
e

molecule
s are

known as the prey
s
. If
the
protein of interest within the bait associates
with the protein found within a prey, the activation and DNA binding domains of the transcription
factor will be

bridge
d, so that reporter gene is

activat
ed
(Fig 7).

The GAL
-
4 is a popular Y2H system.

A
B

C

A

D


Original
Author: R
Appanah




10



multitude of v
ariants of the Y2H exist including
the
one
-
hybrid, three
-
hybrid, reverse two
-
hybrid and
split
-
ubiquitin systems (Hollingsworth & White, 2004).



Fi gure 7: The Y2H system i s based on bai t and prey protei ns. Associ ati on between a prey and the bai t
reconsti tutes

the transcri ption factor acti vity (as the acti vati on and DNA
-
bi ndi ng domai ns are bri dged) whi ch
l eads to the expressi on of reporter genes. Usual l y two reporter
genes are used, one that al l ows a chromogeni c
output

(
e.g.
l acZ

used wi th X
-
Gal ) and one that al l ows

growth sel ecti on (LEU2 or HIS3).

Adapted from
Hol l i ngsworth & Whi te (2004).

3.

Applications of Reporter Gene Systems.

3.1 Monitoring
the
Efficienc
y of G
ene
Transfer
.

The primary usefulness of reporter genes is

to monitor
gene transfer, through a number of
strategies. A
common

example is

the concomitant use of the amp
icil
lin

resistance and lacZ genes (in
plasmids)
-

along with the X
-
Gal substrate
-

to screen for cells which have up
-
taken plasmids
incorporating a certain

gene
of interest. If the gene is present

in the plasmid
, the lacZ is non
-
functional so that X
-
Gal is not cleaved and white colonies are produced. If the gene is absent, the
lacZ is functional so that the X
-
Gal is cleaved to a blue substrate

while if the plasmid

was not taken
up at all, the cells would not be able to grow on
amp
icillin plates. This process is known as the
blue/white colony screening assay (Cronan
et al.

1988).
Another example to illustrate this point is
the use of FACS to detect
the

transfer of G
FP
-
tagged genes in mammalian systems (Mooser
et al.

1997).

Beyond confirming the success of gene transfer, reporter genes are used to monitor the
patterns of expres
sion of tagged genes
.
For example, Chiocchetti
et al.

(1997) have used
GFP
to
monitor gene expression and for promoter mapping in murine models.
Furthermore, a number of
proteins affecting basal and activated transcription levels have been characteri
zed through reporter
gene systems, including interleukins
-
2 and
-
5 (Bamberger
et
al.

1997; Zhang
et al.

1997).

Naylor
(1999) argues that the use of reporter gene systems are particular convenient for monitoring gene
expression as spatial and temporal data of the gene of interest can be obtained
-

even at the level of
singular cells
-

par
ticularly if

the systems are capable of
fluorescent and luminescent output.

1. Library of
‘preys’

2. Recons瑩瑵瑥T
瑲anscrip瑩on
fac瑯r

3. Recons瑩瑵瑥T 瑲anscrip瑩on fac瑯r
ac瑩va
瑥s repor瑥r genes so 瑨a琠
pro瑥in
-
pro瑥in in瑥rac瑩on is
valiTa瑥T.

Original
Author: R
Appanah




11



3.2
Characterizing Protein/Protein and Ligand/Receptor interactions

One of the first protein
-
protei
n
interaction
s

characterized via reporter gene systems was the one
between
the
apoptotic protein p53 and its inhibitor (MdM2) (Momand
et al.

1992). The yeast
-
2
hybrid system
has been extensively utilized

for such characterizations although other rep
orter genes
have been usefully

employed in this enterprise.
In fact, many orphan recep
tors have been cloned
and reporter genes have been used to characterize the
ir

corresponding ligands (Naylor, 1999).
In
fact, r
eporter genes have bee
n
a
particular
ly

attractive
method
to scre
e
n for

the agonis
tic and
antagonistic ligands which bind to

G
-
protein coupled receptors (GPCRs).
Indeed, Stables
et al.

(1997)
have even designed a generic aequorin
-
based reporter assay which is supposedly capable of
characterizing agonist activity at any GPCR.
Furthermore, g
iven the nature of reporter gene system
s,
sophisticated protocols can be designed to investigate complex pathways.

For example,

reporter

gene
s could be used,

with inhibitors of primary effectors
,

to study signalling
pathways and secondary messenger systems by monitoring suspected secondary mess
engers and
effectors.
As an example
,
Ring
et al.

(2011) have devised a complex study involving the use of the
Wnt/Frizzled fusion proteins and the reporter gene HEK 293 cells to study the Wnt signalling
pathway.

3.3 Screening for
Discovery of Novel Drugs a
nd for Toxicological Studies

As stated previously, reporter gene assays can be potently reliable, sensitive and robust. Thus, the
technology is adaptable to HTS studies. HTS for drug discovery using re
porter gene assays is

attractive as the assays are cell
-
based (
Naylor, 1999) so that they

can be monitored over

prolonged
period
s

of time

as the cells grow to confluence and are passaged.
Wood (1995) suggests that,
additionally, reporter gene
-
based HTS studies benefit from th
e possibility of monitoring
system
s

pre
-

(signalling stage) and post
-
gene expression (corresponding to transcriptional and translational
events). Similarly, reporter genes are adaptable to HTS
-
toxicological studies. For example, in the
presence of inhibitors of gene expression, the reporter

signal will cease. Alternatively, truly toxic
substances may perforate cells, liberating the (intracellular) reporter gene from the confines

of the
cell. Imaging would show

decreased intracellular signalling from the reporter with contamination of
culture

medium. In case of extracellular reporter genes

are used
, the reverse will be seen with free
diffusion of the reporter into

the cell post
-
exposure with the toxic agent(s) (Fairey
et al.

1997).
Takahasi & Iwa
h
ashi (
2004) have used GFP
-
based assays to
monitor the expression of 6 different
genes pre
-

and post
-
exposure to 55 chemical entities. They suggest that their assay could be used to
assess and understand the toxicities of newly synthesized chemicals.

4.

Comparing the
usefulness of the

Yeast
-
two
-
hybrid

system and
that of
co
-
immunoprecipitation
to elucidate

protein
-
protein interactions.

Within

cells, proteins are neither geographically nor functionally isolated. Rather, they interact and
associate
with other proteins. P
roteins
also
tend to
have varying l
evels of promiscuity. Thus, to fully
understand of role of a particular protein, its cross
-
talk
associations
with other proteins need to be
characterized

(Markham
et al.

2007)
.
Here
,
the usefulness of the Y2H reporter assay for the study of
such
protein
-
pr
otein

interactions is contrasted to

that of the co
-
immunoprecipitation (Co
-
IP)
assay.
The
overriding
principle behind Co
-
IP is similar to that of the Y2H system and to pull
-
downs methods
Original
Author: R
Appanah




12



(such as the glutathione
-
S
-
transferase pull
-
down) except that the bai
t used is an immunoglobulin
(Stack & Bowie, 2009).

Indeed, Co
-
IP relies on
the dual ‘attachment’ of immobilized
(using matrix beads)
antibodies and of
the unknown prey protein to the bait protein (Fig 8). This procedure allo
ws the easy recovery of the
precipitated complex (unwanted proteins are removed through multiple washing steps). On smaller
scales (such as in laboratory settings), identification of the prey protein is performed through
conventional SDS
-
PAGE and Western blotting. Alternative
ly
, tand
em mass spectrometry
(MS/MS)
or
peptide mass fingerprinting
(PMF)
can be used

for identification of proteins
.
MS/MS is preferred to
PMF when the sample is expected to contain numerous proteins.
A bait exclusion assay (
a repeat of
the experiment without the

antibody) is used as a negative control, ensuring that non
-
specifically
bound protein in the experiment are recognized as such (Markham
et al.

2007).




Fi gure 8: The mul ti
-
step process of co
-
i mmunopreci pi tati on of bai t and prey protei ns

i s depi cted he
re
.

SDS
-
PAGE/Western bl otti ng i s one of numerous downstream protocol s used for i denti fi cati on of the unknown prey
protei n. Indeed, tandem mass spectrometry or pepti de mass fi ngerpri nti ng can al ternati vely be used
for
i denti fi cati on

of the protei ns
.

Image f
rom Acti ve Moti f (nd).

Compared to the Y2H assay, Co
-
IP is a laborious strategy to study protein
-
protein interactions
.
It is
an invasive technique as cells often need to be lyzed or perforated to release the proteins of
interest.
Crude synthesis
of ant
ibod
ies and non
-
specific interaction
s can result in the pull
-
out of
unwanted proteins (Markham
et al.
2007). The process is also lengthy
, particularly if multiple prey
proteins bind specifically to the bait
. However, after isolation, the identity of the prey
protein can be
eloquently assessed through downstream tools (discussed above).

New proteins can be identified
and characterized in this way. These tools tend to be expensive however. On the other hand, Y2H
assays are versatile and cheap. They can be used t
o directly assess
in vivo

protein interactions,
without disturbing the cells.

Moreover, they

can be used for HTS studies, unlike the Co
-
IP (because
of the use of antibodies). However, unknown proteins cannot be directly identified through Y2H
assays. If a
novel protein is suspected, it must first be adequately cloned to be included in the prey
library.
And, o
ften, Y2H systems are not appropriate to study membrane
-
bound proteins and
Original
Author: R
Appanah




13



transcription factors (Hollingsworth & White, 2004).

Finally
, Y2H a
ssays a
re

notorious in that they
have moderate frequencies of false negative and false positive results

(Stellberger
et al.

2010)
.
Interestingly,

Tanowitz
& von Zastrow (2004) have us
ed

bo
th methodologies concomitantly t
o
ide
ntify protein interactions, demonstrat
in
g the
possible complementarity between them.


5.

Critical Appraisal of Research Articles using Reporter Gene Technology.

5.
1
Elaboration of a monocistronic dual reporter to evaluate the replication of
the
hepatitis C virus.


[
A
Critical Appraisal of “A novel luciferase and GFP dual reporter virus for rapid and convenient
evaluation of hepatitis C replication” by Wu
et al.

(2011).]

The hepatitis C virus (HCV
) is an important human pathogen which

was only identified in 1989
through molecular isolation (Choo
et al.

1989; Houghton, 2009)
. Globally, up to 180 million people
are believed to be infected and the disease primarily affects the liver, leading to asymptomatic liver
sca
rring and may ultimate
ly result in

cirrhosis and hepatic carcinomas (Rosen, 2011).

Frequently, the
only way to diagnose chronic
H
C
V

infections is through liver biopsies

which are costly and which can
lead to complications. Although a number of treatment strategies are available

-

including
interferon
-
based antiviral therapy (such as pegylated INF
α
) and antiviral agents (for example
ribavirin
, telaprevir and boceprevir
)
-

no
vaccine against

H
C
V

has been developed yet

(Rosen, 2011;
Wilkins
et al.

2010;
Wu
et al.

2011).
This is

worr
ying as

the transmission routes of H
C
V
, such as

intravenous drug use
, are very difficult to control.
T
he elaboration of specific antiviral agents and
vaccines
against the H
C
V

has been hampered by the dearth
of a full viral

life cycle cell culture system

(W
u
et al.

2011).
However, in 2005, three research groups have

managed to produce infectious H
C
V

particles by transfecting Huh
-
7 (hepatoma
-
derived) cell
-
lines (or derivatives thereof) with the JFH1
viral genome (Lindenbach
et al.

2005; Wakita
et al.

2005; Zhong
et al.

2005).
Despite such advances
however, the protocols used to quantify HCV infection and replication in those cell cultures are
exceedingly laborious (Wu
et al.

2011).

Dedicated single reporter gen
es (including GFP and
luciferase) have be
en constructed, but these have not been popular in the research community for
antiviral drug screening.

This is because while GFP reporter genes can be directly visualized through fluorescence, the precise
quantification of
this
fluorescence remains labor
ious and time
-
c
onsuming. Furthermore, the
background

in GFP assays is too high, leading to relatively poor contrast. Likewise, single luciferase
gene reporters are sensitive and easily performed, but cannot be directly visualized

(Wu
et al.

2011)
.
Bicistro
nic
dual
repor
ter constructs have been at
te
mpte
d but these increased the size and
complexity of the HCV genome beyond 12,000 nucleotides, impeding the repli
cation process
(
Dansako
et al.

2008)
.
Thus, Wu
et al.

(2011) planned to construct a dual
, monocistro
nic

reporter
gene for HCV, composed of GFP and luciferase so as to exploit the visibility of the former and the
quantifiability of the latter.
This approach
had successful antecedents

as Wang
et al.

(1999) have
demonstrated that GFP and luciferases fusion
constructs
were possible and that the individual
components
did not
significantly
interfere with each other’s signal.

The authors used the cell
-
line
Huh
-
7.5.1
, similar to

Zhong
et al.

(2005
)
,

which

was

experimentally convenient
as
Huh cell
-
lines are

an
established
culture systems for HC
V

(Duverlie & Wychowski, 2007; Lindenbach
et al.

2005)
.
The
first part of the experiment dealt with construction of the dual reporter and assessing its effects on
the HCV (summa
rized in Fig 9). T
he reporter was
then
used t
o
monitor

effects of antivirals on HCV.


Original
Author: R
Appanah




14





Fi gure 9:

Thi s i mage depi cts the

i ni ti al stage
s

of the study, i ncl udi ng: (1) the producti on of the dual reporter
construct (red and bl ue),
al ong wi th si ngl e reporter (red or

green), repl i cati on i ncompetent (star
) and wi l d
-
ty
pe
pl asmi d constructs
; (2,3)
the
in vitro

transcri pti on and
subsequent
transfecti on of Huh cel l s wi th transcri pts
through
el ectroporati on
. Later stages

(4
-
8)

are al so summari zed
.

Image constructed usi ng

Powerpoi nt.

The authors used a plasmid
encoding the complete genomic sequence of a HCV genotype
(JFH1)
and
proceeded to make single reporter constructs (
of GFP or

luciferase),

a monocistronic GFP/
lucifera
s
e
-
dual reporter construct and a replicative
-
incompetent construct.
The
GFP was linked to a

sequence
coding for a protein (NS5A).
The
replicative
-
incompetent

construct would be used as a negative
control and was constructed through the introduction of a point mutation which inhibits RNA
polymerase activity. Wild
-
type plasmids
were

used as positi
ve controls. Although the identities of all
the constructs were confirmed through sequencing, their usefulness need
ed

to be assessed. For this
purpose, the plasmids were linearized and used as template for transcript production. Optical
density measurement
s at 260 nm and agarose gel electrophoresis were used to assess the
concentration and integrity of the RNA molecules post
-
transcription. However, it is felt t
hat the
authors sh
ould have used the ratio of OD
260
/OD
280

(OD: optical density)
to
compute

the ext
ent of

DNA
-
mediated contamination

(Yang

et al.

2008
).

This is because the following

step in the
experiment involved the introduction of the RNA into Huh
-
7.5.1 cells through electroporation
.
Simultaneous entry of contaminant DNA would affect the quantity of transcripts (and derived
measurements) within
the
cells
.
Electroporation
is
an
adequate
tool for transfection
as hep
atocytes
adjust competently to the procedure

(Yao
et al.

2002), with hi
gh survival rates. However, it is not
understood why the authors preferred to introduce RNA rather than the plasmid constructs directly
into the Huh cells. Perhaps transfection of the mRNA was easier than that of the plasmids, but this
was not mentioned by

the authors, nor is it supported by any literature. Indeed, the
in vitro

transcription seems like an unnecessary step.


Original
Author: R
Appanah




15



Five days post
-
transfection, Huh
-
cells were lyzed and
analysed through Western blotting
and
indirect immu
no
fluorescenc
e assay (IFA/ confocal microscopy)
to determine the viral protein
compositions of the samples.
For the IFA protocol, the samples were washed with PBS between
incubation periods of primary and secondary antibodies. It is felt that PBS is not strong enough a

detergent which may lead to pronounced background staining. Instead, Triton
-
X 100 could be used
for the

washing
s
teps
.
However, the IFA experiment was successful as GFP was shown to co
-
localize
with NS5A
in the dual reporter construct
(Fig 10.A).

This was

validated by western blot which
detected a

NS5A
-
GFP fusion protein

for both the dual construct and the single GFP reporter. Hence,
GFP is a valid marker of viral replication loci. T
he luciferase did not perturb this fusion protein in the
dual reporter con
struct.
After transfection, s
ome

Huh

cells were grown so that they experienced
cytopathic effects due to the continuous viral infection. The supernatants were used to calculate the
viral titre levels while the remaining cells were used for luciferase assay studies and to calculate the
intrac
ellular concentration of HCV RNA (measured via RT
-
qPCR). The titres and quantity of RNA were
seen to correlate
and the dual reporter construct did
not
mediate significant decreases in levels of
viral

RNA or in viral titre levels
, compared to the singular r
eporter constructs and to the positive
control
.

The luciferase activity of the dual reporter was comparable to that of single Luc reporter (Fig
10.B).

In fact, after infecting naïve Huh
-
7.5.1 cells with the dual reporter construct, Wu
et al.

(2011)
observ
ed a time
-
dependent increase of luciferase activity and GFP fluorescence intensity which
correlate
d

with

both

the
infectivity and RNA levels

of HCV (Fig 10.C)
.





Fi gure 10. A:

Herei n, GFP was experi mental l y shown to co
-
l ocal ize wi th NS5A wi thi n i nfected cel l s

(i n both
si ngl e and dual reporter constructs)
. B: The l uci ferase acti vi ty
of the

dual construct was si mi lar to that of the
si ngl e Luc
-
reporter construct. C: The
useful ness

of the dual reporter was tested i n Huh cel l s. The l evel of HCV
RNA and i nfecti vi ty of the vi ral RNA was found to correl ate el egantl y wi th the l uci ferase acti vity and

the

number of fl uorescentl y
-
l i t l oci (by vi rtue of GFP).

Image adapted from Wu
et al.

(20
11).

A

B

C

Dual reporter

Single
-
Luc
reporter

Original
Author: R
Appanah




16



The authors also investigated the usefulness of this novel dual reporter to monitor the effects of
antivirals on the viability of HCV in infected cells. For this purpose, they pre
-
treated Huh cells with
either IFN
α
-
2b and ribavirin,
as these two
agents are frequently used to treat HCV infections
.

IFNα
-
2b is
also
anecdotally recognized as the more potent variant of the substance

(
Houghton, 2009)
. The
results were very encouraging (Fig 11
.A
-
B
), showing a dose
-
dependent decrease in the activity of
luci
ferase and GFP for both agents.





Fi gure 11. The dual reporter was used to i nvesti gate the
dose
-
dependent
effects of anti vi rals (A) IFN
α
-
2b and
(B) ri bavi rin on the abi l i ty of HCV to sustai n i ts i nfecti
on i n Huh
-
7.5.1 cel l s. D
ecr
eases i n the l evel s of vi ral RNA
correl ated wi th

decreased fl uorescence at NS5A l oci (decreased repl i cation) and decreased l uci ferase acti vi ty.
Thus, thi s dual reporter coul d be used for hi gh
-
throughput screeni ng of
therapeuti c agents agai nst

HCV. Image
ad
apted

from Wu
et al.

(2011).

This
study was

very well thought
-
out and the

paper

was
equally very well
-
written
, although possible
improvements

to the protocols have been suggested. The diagrams conveyed information well

although
the use of
colour
ed

lines

in the graphs

would have enhanced
the ease of data
interpretation. Moreover,

some of the figure legends were somewhat confusing.
Yet
, Wu
et

al.
(2011)
have devised a highly

potent tool which allows the visualisation of
viral replication (through
GFP
-
media
ted fluorescence) and quantification of rates of inhibition
of HCV replication
by
monitoring the levels of luciferase activity. Importantly, this dual reporter does not significantly
affect the viability and infecti
vity of

HCV
. Additionally, by virtue of m
onitoring GFP
-
mediated
fluorescence, the optimum measurable values of luciferase activity can be anticipated, reducing the
risk of empirical failure (Wu
et al.

2011). The ability and sensitivity of this tool to detect inhibition of
viral replication (Fig 1
1.A
-
B) will be useful to screen for potential

agents to treat or cure HCV
infections
, particularly since this technique is amenable to high
-
throughput studies.





A

B

Original
Author: R
Appanah




17



5.2
Using luciferase reporter gene assays to characterize the gene expression of
the
two
mini open
reading frames (uORFs) found in the obscure p13 gene in

the

Helicoverpa armigera

nucleopolyhedrovirus
.

[A Critical Appraisal of “
Rare codons in uORFs of baculovirus
p13

gene modula
tes downstream gene
expression” by
Qiao
et al.

(2011)
]

Nucleopolyh
edroviruses (N
PVs) represent a genus from the

Baculoviridae

family

and can be sub
-
divided in groups I and II, according to their molecular phylogenities particularly their nucleoplasmid
per virion ratio (Herniou
et al.

2003).
NPVs affect the Lepidoptera or
der of insects and have been
envisaged as biological agents against pest
s
, thus generating financial interest.
Group II NPVs (such
as
Leucania separata

and
Helicoverpa armigera

NPVs
) and Granuloviruses (another genus of
Baculoviridae
) are the only organism
s known to

express the
p13

gene. In

the

Leucania
separata
NPV,
the
gene encodes a glycotransferase

(Ls
-
P13)

(Christopher & Stephen, 1997) while promoter analysis
has shown that
it

ambiguously incorporates both early (CAT/AT) and late (TTAAG) promoter motifs
(Fig 12)
(Qiao
et al.
2011
). Moreover, recombinant
Autographa californica

NPV expressing the P13
protein
from

the

Leucania separata

NPV
displayed
a
decreased yield of polyhedral
s
, but conversely,
an
increased yield of budded virions on infection
of
insect Sf9 cells (Du
et al.

2007).
The larval
-
killing
pote
ntial of this

recombinant
Autographa californica

is
also
enhanced compared to wild
-
type.
Du
et
al.

(2007) have also shown that

Ls
-
P13 is a transmembrane protein
which locates in the host’s
nucleus on infection, but it relocalizes and incorporates itself into the host’s cytoplasmic membrane
48 hours after infection. Consequently, the
p13

gene
must encode for diverse (and perhaps,
conflicting) instructions which need to be efficiently regulated.


Fi gure 12: The earl y (enci rcl ed i n orange) and the l ate (enci rcl ed i n red) promoter sequences i n the
p13

gene,
found 294 and 17 bases upstream of the transcri pti on start site, respecti vel y. Qi ao
et al.

(2011).

It is believed that

the complex

modulation of the activity of
the
p13

gene is possible because of the
presence of rare codons and
mini open reading fr
ames (uORFs) encoded with
in

the
p13

gene itself
(Fig 13
.A
).
uORFs have been identified in

the

5’ UTR
(un
-
translated region)
of

viral genes (e.g the
Human Immunodeficiency Virus and the Hepatitis B virus)
and within eukaryotic genes (Chen
et al.

2005; Krumm
heuer
et al.

2007).

Such uORFs are known to
regulate the expression of the
ir

main
,
downstream

open reading frame

(through a number of possible fates) (Fig 13.B)
, although this
influence is flexible and can either promote or stifle gene expression (Harding
et al.

2000; Meijer &
Thomas, 2002; Morris & Geballe, 2000).
Thus, to understand the function of the two mini ORFs
within the
p13

gene, Qiao
et al.

(2011) have designed a l
uciferase
-
based study using the
Helicoverpa
armigera

NPV.

This NPV (as it name indic
ates) infects
Helicoverpa armigera
(cotton bollworm) and
has been
utilized
as a biological agent.



Original
Author: R
Appanah




18







Fi gure 13. A: The two mi ni ORFs (doubl e
-
arrowed) found upstream of the
p13

gene

(an al ternati ve vi ew of
Fi gure 12). Image taken from Qi ao
et al.

(2011). B:
The al ternati ve fates possi ble to a ri bosome after transl ating
a uORF. The ri bosome
-
mRNA hybri di zati on can be undi sturbed wi th transl ati on resumed at ei ther a proxi mal
(opti on 1)
or a di stal AUG (opti on 2), hence medi ati ng no effect on downstream transl ati on. Another
al ternati ve avai l able (opti on 3) i s the stal l ing of the ri bosome duri ng ei ther the el ongati on or termi nati on
phase of the mi ni ORF transl ation. Thi s bl ocks additi onal

ri bosome acti vi ty, i nhi biti ng downstream gene
expressi on. uORFs may
al so di rectl y i nfl uence gene expressi on through al teri ng the
stabi l ity

of transcri pts
(opti on 4). Fi nal l y, the ri bosome may fai l to di ssociate after transl ation of the uORF, affecti ng dow
nstream
transl ati on (opti on 5) (Morri s & Gebal l e, 2002).

Opti ons 3 and 5 are subtl y di fferent.

In the study,
Qiao
et al.

(2011) engineered novel l
uciferases constructs

to investigate the effects of
uORFs and rare codons on the gene expression of the main O
RF

(Fig 14. A,

C,

E). For this purpose,
the plasmid vector pGL3 encoding the firefly (
Photinus pyralis
) luciferase was utilized (Promega).
However, this plasmid is used in tandem with
Renilla

luciferase pRL vectors which are constitutively
active, providin
g an internal control

(Promega, 2012)
. pRL vectors however are ill
-
adapted for
transfection in insect cells, so that Qiao
et al.

(2011)

had to engineer

a novel plasmid vector (pIZ/V5
-
His
-
hRL)
which has been found to be stable and

a highly efficient internal control to the pGL3
-
luc in
the Hz
-
AMI
insect
cell
-
line

used in the experiment
. This invention will be of outstanding value to the
scientific community (as an internal control to firefly luciferases) if it is found to be stable
in a variety
of
other
insect cell
-
lines and primary cell cultures. The production of different plasmid constructs
were adequately described

in the text
, including the use of endonucleases (such as HindIII)
and
primers.
The constructs were also detailed gra
phically in sufficient detail (Fig 14. A, C, E).
Unfortunately, h
owever, the authors fail
ed

to describe how they induced mut
ations in some of the
plasmids. T
he authors
sequentially
co
-
transfected
Hz
-
AMI
cells with the
novel
pGL3
-
Luc

construct
s

and the inte
rnal control,

pIZ/V5
-
His
-
hRL (using 1.0
µ
g of each vector per assay). Each experiment
was conducted in triplicate (a widely accepted number of replicates for cell
-
studies). 48 hours after
co
-
transfection, the
activities of the firefly and
Renilla

luciferas
es were assessed using the Turner
Designs TD
-
20/20, a sensitive luminometer which has been extensively used for luciferase
-
based
studies (Anderson
et al.

2008; Sugawara & Takeuchi, 1997). The results were expressed as a ratio:
















































The luciferase activity ratios are represente
d grap
hically in Figures 14. (B, D,

F). However, the
authors did not indicate how they calculate
d

the values for the error bars in the luciferase activity
ratio assays plots depicted below. Presumably, for each ratio, they
had to calculate

the standard

uORF

A

B

Original
Author: R
Appanah




19



deviations for both the luciferases from their triplicates and computed the respective errors from
the
se

paired standard deviations. Although this is a minor omi
ssion, it

brings uncertainty to the

ex
tent of error within replicates and, thus, to the reprodu
cibility of the experiment.











Fi gure 14.
A: The pWt
(‘wi l d
-
type’)
, F1 and F2
construct
s

used to assess the extent of gene expressi on of the
mai n ORF, of uORF1 and uORF2 (of the
p13

gene), respecti vel y. B: The l uci ferase acti vity profi l es
of the pWt,
F1, F2 constructs (a
l ong wi th a negati ve control
, ‘Mock’
) 48 hours after transfecti
on i n Hz
-
AMI cel l s. C: The pWt
was used to desi gn constructs wi th mutant (and, hence, non
-
functi onal ) start co
dons of uORFs. The

l uci ferase
acti vi ty profi l es
of these constructs,
48 hours post
-
transfecti on
, are depi cted i n (D)
. Si mi l arly, (E) shows the
con
structs
wi th substi tuted rare codons whi l e

thei r correspondi ng l uci ferase profi les 48 hours post
-
transfecti on i s shown i n (F). In both (C) and (E)
, the abbrevi ated names of the constructs (boxed) are shown
adjacent to them (to the l eft) and the
mutated or
substi tuted
codons or depi cted by arrows. Image adapted
f
rom Qi ao
et al.

(2011).

Initially, the authors constructed
a
plasmid whereby the luciferase was attached to the main ORF of
the

Helicoverpa armigera

p13

gene (pWt) (Fig 14. A, C, E). This would act a
s a positive control for the
entire experiment. Plasmids with luciferase attached to uORF1 (the most upstream mini ORF) and
with luciferase attached to uORF2 was constructed

(labelled F1 a
nd F2, respectively, in Fig 14.A
). This
was done by removing the sto
p codon of the uORFs and the start codon of luciferase. This was an
pWt

pWt

Mock

F1

F2

F1

F2

C

L1

L3

pWt

Mock

L2

E

C2

C1

C3

pWt

Mock

A

F

D

B
A

Original
Author: R
Appanah




20



ingenious step as the uORFs and the luciferase would be translated as one protein. Although
unmentioned by the authors, this step ensures that stability issues inherent to the
transcripts
and
proteins are not problematic. Had the authors not removed these stop and start codons, the uORF
s

and
the
luciferase would have been translated stoichiometrically, but they could be digested at
different rates,
potentially
leading to disparate quantitie
s of the uORF gene product and luciferase

at
various time
-
points
.

The same

reasoning applies to the mRNA.

The pWt, F1 and F2 vectors (along with a mock, representing a negative control) were co
-
transfected
individually with the internal control in
Hz
-
AMI
cells. 48 hours post
-
transfection, the expressions of
the reporter genes were a
ssayed and expressed as ratios

(Fig 14.B).
Both vectors expressed the
reporter gene

which was a success in itself, validating the feasibility of the author’s approach to
study m
ultiple mini ORFs in genes where they occur. The F2 and F1 constructs expressed greater
firefly luciferase activity than pWt, and the activity of the F2 construct was much greater than that of
the F1. Qiao
et al.

(2011) interpreted this as meaning that the

uORFs with longer sequences are
more successful in translational activation. This is not supported by other scientific publications and
is not a reasonable claim. In fact, information encoded within the ORFs

(and not nucleotide length)

may be responsible
for differences in the extent of translational activation.

The authors then sought
to understand the effect
s

of the mini ORFs on the gene expression of the main ORF. For this
purpose, they introduced point mutation in the pWt construct at the start codon o
f
uORF1 (L1), at
the start codon of uORF2 (L2) and simultaneous
ly

at both start codons (L3)

(Fig 14.C)
. These
constructs along with the positive and negative controls were introduced in
Hz
-
AMI cells and the
cells were assayed 48 hours later. The results (F
ig 14.D) demonstrated that gene expression of the
main ORF was highest in the L3 construct and next
-
highest in the L1 construct. The gene expression
in the L2 construct was still about four times as high as observed in the pWt, but it was substantially
les
s than in the L1 and L3 constructs. The authors correctly point
ed

out that this correlates with the
uORF1 being a stronger inhibitor of downstream

gene expression

compared to

uORF2, and that
these two mini open reading frames may collaborate during gene re
gulation of the main ORF. The
authors also marvel
at the fact
that the length of the uORF does not
determine its strength as an
inhi
bitor of translation. It is felt that the authors believed that the complexity of a

uORF
correlated
with its length, which i
s not necessarily true (as observed herein).


Finally, Qiao
et al.

(2011) determined the effects of rare codons
embedded in the uORFs. Comparing
the codons found in the
uORFs of the
p13

gene of the
Helicoverpa armigera

NPV

with those found in
the
Baculovirus polyhedrin genes and insect genes, they
found one rare codon (AGA) in the uORF1
and two rare codons (ACA and TCA) in the uORF2 (Table 2).
The authors comment
ed

that
codons
which are effectively rare in genomes, occur at

ambiguously

high frequen
cies in the uORFs of the
p13

gene.
Whether this is an evolutionary conserved feature of uORFs remains to be seen.
Qiao
et
al.

(2011) proceeded to design constructs where either the rare codon in uORF1 was (C1) or the rare
codons in uORF2 (C2) or all three
rare codons (C3) were substituted with more common codons,
exploiting the degeneracy of the genetic code (Fig 14.E). Again the constructs were co
-
transfected
individually with the pIZ/V5
-
His
-
hRL in the Hz
-
AMI cell
-
culture, and the cells were assayed 48 hou
rs
post
-
transfection.



Original
Author: R
Appanah




21



Tabl e

2
: The three rare codons found by the authors i n the mi ni ORFs of the
p13

gene, and thei r rel ati v
e
frequenci es across di fferent genes.


AGA

ACA

TCA

Baculovirus polyhedron
genes

0.9%

0.5%

0.3%

Insect genes

0.5%

0.9%

0.9%

uORF
s

from the p13 gene

1.0%

2.0%

4.1%


The results (Fig 14.F)
demonstrate
d

that the rare codon in the uORF1 is more competent in inhibiting
downstream gene expression and the two rare codons found in the uORF2. Moreover, substituting
all three rare codons results in the greatest increase in the firefly luciferase activity and, h
ence, in
downstream gene expression. The authors
however fail to remark that the increases in gene
expressions after substituting rare codons were greater than the corresponding mutagenesis of the
uORFs
’ start codons

(comparing Fig 14.D and 14.F). Hence, w
e can conclude that the rare codons
within the uORFs might be more

competent

for

the inhibition of downstream gene expression than
the full
-
length uORF
s
themselves
. However, we cannot suggest a v
alid theorem to complement this
observation
.

In conclusion,
Qiao
et al.

(2011) have penned

a very good paper with
an excellent

standard of
English. The paper is well
-
referenced, although it is slightly concerning that about half of the papers
quoted are previous works of the authors themselves. Still, the experimen
t design was excellent,
although some of the claims made by the authors (mainly in the results section) are speculative at
best. The overall organisation of figures was not judicious (although it is appreciated that this is not
the fault of the authors). T
he bar
-
charts were ambiguously labelled numerically (1 to 5)
. Although
the figure legends provided a clear indication of the samples these numbers corresponded to,
directly labelling the bar
-
charts with the samples tested (or an appropriate abbreviation th
ereof)
would have greatly improved the ease in interpreting the data (
as done in
F
ig 14. B, D,

F).
Meanwhile, t
he outcomes of this paper are expansive. Firstly, a novel

insect
-
specific
Renilla

vector
(to be used as an internal control to firefly luciferase

vectors) has been designed, and this approach
to study uORFs may be replicated elsewhere, because of its elegance

and simplicity. The insights on

the importance of uORFs and of rare codons incorporated in them afforded by this paper may also
be useful in
molecular biology. We suspect that the immediate impact of this paper would be
based
on

devising

recombinant biological agents against pests.



Original
Author: R
Appanah




22



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