Báo cáo khoa học: Characterization of the Drosophila Methoprene -tolerant gene product Juvenile hormone binding and ligand-dependent gene regulation potx

Characterization of the Drosophila Methoprene -tolerant
gene product
Juvenile hormone binding and ligand-dependent gene regulation
Ken Miura, Masahito Oda, Sumiko Makita and Yasuo Chinzei
Department of Medical Zoology, School of Medicine, Mie University, Tsu City, Japan
Insect development and reproduction are regulated by
two classes of lipid-soluble hormones, the ecdysteroids
and juvenile hormones (JHs). The ecdysteroids activate
target genes through a heterodimeric receptor complex
composing the ecdysone receptor and ultraspiracle
(USP) proteins, both of which are members of the nuc-
lear steroid ⁄ thyroid ⁄ retinoid receptor superfamily [1].
During insect development, ecdysteroids induce molting
while JH determines the nature of each molt by modu-
lating the ecdysteroid-induced gene expression cascade
[2–4]. In addition, in adult insects, JH has a wide
variety of actions related to reproduction, including
oogenesis, migratory behaviour and diapause [2,5,6].
The mode of molecular action of JH, however, is still
obscure [7]. JHs are a family of esterified sesquiterpe-
noids, whose lipid-soluble nature has suggested action
directly on the genome through nuclear receptors such
as ecdysteroids and the vertebrate steroid ⁄ thyroid ⁄ reti-
noid hormones [5,8] although actions of JH through
the cell membrane are also documented [9,10].
Many attempts have been made to identify nuclear
JH receptors. Jones and Sharp [11] showed that JH III
binds to the Drosophila USP protein, which is a homo-
logue of the vertebrate retinoid X receptor, promoting
Keywords
juvenile hormone; juvenile hormone

receptor; Methoprene-tolerant; Drosophila;
transcription factor
Correspondence
K. Miura, Department of Medical Zoology,
School of Medicine, Mie University,
Edobashi 2-174, Tsu514-8507, Japan
Fax: +81 59 231 5215
Tel: +81 59 231 5013
E-mail:
(Received 27 October 2004, revised 20
December 2004, accepted 4 January 2005)
doi:10.1111/j.1742-4658.2005.04552.x
Juvenile hormones (JHs) of insects are sesquiterpenoids that regulate a
great diversity of processes in development and reproduction. As yet the
molecular modes of action of JH are poorly understood. The Methoprene-
tolerant (Met) gene of Drosophila melanogaster has been found to be
responsible for resistance to a JH analogue (JHA) insecticide, methoprene.
Previous studies on Met have implicated its involvement in JH signaling,
although direct evidence is lacking. We have now examined the product of
Met (MET) in terms of its binding to JH and ligand-dependent gene regu-
lation. In vitro synthesized MET directly bound to JH III with high affinity
(K
d
¼ 5.3 ± 1.5 nm, mean ± SD), consistent with the physiological JH
concentration. In transient transfection assays using Drosophila S2 cells
the yeast GAL4-DNA binding domain fused to MET exerted JH- or JHA-
dependent activation of a reporter gene. Activation of the reporter gene
was highly JH- or JHA-specific with the order of effectiveness:
JH III  JH II > JH I > methoprene; compounds which are only structur-
ally related to JH or JHA did not induce any activation. Localization of

MET in the S2 cells was nuclear irrespective of the presence or absence of
JH. These results suggest that MET may function as a JH-dependent tran-
scription factor.
Abbreviations
Ahr, aryl hydrocarbon receptor; Arnt, Ahr nuclear translocator; bHLH, basic helix-loop-helix; DBD, DNA binding domain; DCC, dextran-coated
charcoal; EGFP, enhanced green fluorescent protein; JH, juvenile hormone; JHA, synthetic analogue of JH; Met, Methoprene-tolerant gene;
MET, Met protein; PAS, period-aryl hydrocarbon receptor ⁄ aryl hydrocarbon receptor nuclear translocator-single-minded; Per, Drosophila
period clock protein; Sim, Drosophila single-minded protein; SFM, serum-free medium; TNT, coupled in vitro transcription ⁄ translation;
UAS, upstream activating sequence; USP, ultraspiracle protein.
FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS 1169
its homodimerization, but the concentrations of JH
required are several orders of magnitude higher than
its physiological titre [12]. In transiently transfected
cultured cells, a high dose of JH led to transcriptional
activation through the binding of USP to a DNA
response element upstream of a core promoter [13]. In
other insect species, gene regulation by JH through
DNA sequences resembling nuclear hormone response
elements has been reported [14–16], suggesting the
involvement of nuclear receptor family members in JH
signaling.
Because the strict regulation of JH titre in the insect
body is crucial [17], the application of exogenous JH
or analogues (JHAs) can disrupt normal development,
and a number of JHAs have been synthesized and used
as insecticides as well as research tools. The genetic
and biochemical studies on resistance to JHA insecti-
cides have led to the implication of another class of
transcriptional regulator in JH signaling. Wilson and
coworkers examined the resistance mechanism of Dro-

sophila to the JHA, methoprene, and isolated mutant
lines of flies that are resistant to morphogenetic and
lethal effects of natural JH or methoprene [18]. The
allele responsible for this resistance, named Metho-
prene-tolerant (Met), encodes a basic helix-loop-helix
(bHLH)-PAS protein, MET [19]. The bHLH-PAS fam-
ily comprises transcriptional regulator proteins that are
key players in a wide array of developmental and
physiological pathways such as neurogenesis, circadian
rhythms, hypoxia response, and toxin metabolism
[20,21]. PAS is an acronym from the initial members
of the family: Drosophila period clock protein (Per),
vertebrate aryl hydrocarbon receptor (Ahr, also known
as dioxin receptor) ⁄ Ahr nuclear translocator (Arnt),
and Drosophila single-minded protein (Sim) [22,23].
The bHLH-PAS transcription factors share a com-
mon overall structure. The bHLH domain is located
near the N terminus. The basic region binds to a con-
sensus palindromic hexanucleotide E-box (CANNTG)
[24] or its derivatives [25,26]. The HLH domain allows
these proteins to form a hetero- or homodimer. The
bHLH domain is followed by PAS-1 and PAS-2
domains, which are used for dimerization between PAS
proteins, small molecule binding, and also for binding
to non-PAS proteins. The C-terminal half residues,
which are not well conserved, harbour transcription
activation ⁄ repression domains [20]. These structural
features are found in MET [19]. Met mutant flies exhi-
bit low JH binding affinity in fat body cytosolic
extracts while an 85-kDa protein seems to be respon-

sible for this binding [27,28]. Localization of MET in
Drosophila tissues is exclusively nuclear [29]. Met
females show reduced oogenesis [30], and the males
have some defects in reproduction [28,31]. The Met null
mutant flies are viable, showing that Met is not a vital
gene, but this might be explained by redundancy provi-
ded by cognate genes [30]. These observations suggest
the involvement of MET in at least one pathway of JH
signaling. The direct evidence, however, is still lacking:
does JH bind directly to MET?; does MET function as
a JH-dependent transcriptional regulator?
We have now examined the binding of radiolabeled
JH III to MET protein. Using a heterologous system
in Drosophila S2 cells, we have characterized ligand-
dependent gene regulation by MET. Our results
suggest that MET may function as a JH-dependent
transcription factor.
Results
MET binds to JH III with high affinity
The MET protein was obtained by using coupled
in vitro transcription ⁄ translation (TNT) reaction. The
production of the full-length polypeptide was confirmed
by analysing the product of reaction in the presence of
35
S-methionine by SDS ⁄ PAGE and autoradiography
(Fig. 1). As a negative control, mock-programmed
lysate was processed in parallel. As evident in the figure,
the principal product had the expected full-length
molecular mass of 79 kDa. Faster migrating minor pro-
tein bands are also visible, which were not eliminated by

the addition of protease inhibitor mixture in the reac-
tion (data not shown). Then, the programmed lysate
was used as a protein source for binding assay by the
dextran-coated charcoal (DCC) method. As seen in
Fig. 2, specific binding of
3
H-labeled JH III to the TNT
protein showed saturable profile. The experiment was
repeated three times, and the K
d
value by Scatchard
analysis was calculated to be 5.3 ± 1.5 nm (mean ±
SD). By these experiments, it was demonstrated that
MET binds to JH III directly with a nanomolar K
d
value although we do not rule out the possibility that
factors in the rabbit reticulocyte lysate may influence
binding. The specific binding was competed away by
100-fold molar excess of cold JH III (data not shown).
MET regulates transcription in a JH-dependent
manner
The Met gene product was examined for its transacti-
vation ability. The binding sequence motif of MET is
presently uncertain. In addition, it is unknown whether
MET functions as a homo- or heterodimer. So, we
utilized a heterologous approach with the yeast
GAL4–DBD (DNA binding domain) fusion ⁄ UAS
Characterization of Drosophila Met gene product K. Miura et al.
1170 FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS
(upstream activating sequence) system. The GAL4–

DBD possesses a zinc finger that directs homodimeri-
zation and binding to UAS elements, and a potent
nuclear localization sequence [32,33]. By using this
system, we were able to assess the transactivation
potential of MET independently of its dimerization
properties or nuclear localization signals. The MET
protein was expressed in S2 cells as a fusion with
GAL4–DBD, and a luciferase reporter construct pos-
sessing five tandem copies of the UAS in its regulatory
region was used. In this system, the effect of JH on
transcription from the reporter gene was tested.
As shown in Fig. 3, when an empty expression vec-
tor was transfected, the addition of 5 lm JH III to the
culture medium caused no elevation of reporter activ-
ity over that of controls given the vehicle ethanol.
Expression of native MET lacking GAL4–DBD
slightly elevated the reporter activity, but did not show
any JH dependency. Next, only the GAL4–DBD was
expressed. In this case, the reporter activity was eleva-
ted about twofold above the empty vector control in
either the presence or absence of JH III, indicating
that the GAL4–DBD translocates into the nucleus and
functions as a moderate, constitutive activator of
transcription in a JH-independent manner. The
GAL4–DBD–MET fusion in the presence of ethanol
did not bring about any enhanced reporter activity
relative to the empty vector control, but when JH III
was added, the reporter activity was elevated about
fivefold over the case of the empty vector control or
the case of the GAL4–DBD–MET with ethanol. This

activation by JH III can also be described as about
twofold when compared to the case of GAL4–DBD
with JH III. This indicates that MET has transactiva-
tion domain(s), and its transactivation function is JH
dependent. It is noteworthy that in the absence of
JH III the MET moiety of the fusion protein repressed
the moderate transactivation produced by GAL4–
DBD. This suggests that unliganded MET may func-
tion as a transcriptional repressor.
Fig. 1. Autoradiogram of TNT lysate programmed with Met cDNA.
The TNT reaction was performed with 400 ng PCR fragment con-
taining T7 promoter and Met full ORF in the presence of
35
S-methio-
nine. A portion of the lysate was separated by 10% SDS ⁄ PAGE
and autoradiographed. A mock-programmed lysate was run in paral-
lel. Molecular mass markers are shown at the left. An arrowhead
indicates the position of full-length MET (79 kDa).
Fig. 2. MET binds to JH III with high affinity. (A) Binding of
3
H-labe-
led JH III to MET. MET was obtained by TNT reaction and subjec-
ted to DCC assay with
3
H-labeled JH III as described in
Experimental procedures. Specific binding was calculated by sub-
tracting the counts of mock-programmed lysates from those of cor-
responding programmed lysates. The specific binding is shown in
the figure. (B) Scatchard analysis of JH III binding to MET. The K
d

value was calculated from the slope of the regression line. These
analyses were performed three times and representative data are
shown.
K. Miura et al. Characterization of Drosophila Met gene product
FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS 1171
Ligand specificity of transactivation by MET
If MET represents a JH-dependent transcription fac-
tor, it should show stringent ligand specificity. To test
this, several compounds that are structurally related to
JH or JHA but show no JH activity were examined in
the GAL4-MET fusion ⁄ UAS system. The effects of
these potential ligands on the reporter activity are
shown as fold induction by dividing the activity
obtained with the pAcGAL4–DBD–Met by that in
negative controls using empty pAc vectors (Fig. 4). As
is evident here, addition of squalene, farnesol, farnesyl
acetate and geraniol at a final concentration of 5 lm
did not result in any activation of the reporter. JH III,
however, again brought about enhanced reporter
activity. Interestingly, a JHA ) methoprene ) showed
weaker ligand activity than JH III. Thus, the trans-
activation exerted by MET shows stringent ligand
specificity apparently related to JH activity, ruling out
nonspecific transactivation by lipid-soluble compounds.
Dose–responses of natural JHs and JHA on MET
transactivation
The binding assay showed that MET has a nanomolar
level K
d
for JH III and we used several potential lig-

ands at 5 lm in the experiments described above. If
MET functions as a JH-dependent transcription factor,
it should respond to nanomolar levels of ligand, con-
sistent with its high affinity for JH III. Here, we tested
three natural JHs, JH I, JH II, and JH III, and the
JHA methoprene in varying concentrations using the
GAL4-MET fusion ⁄ UAS transfection assay (Fig. 5).
The effects on the reporter activity are shown as fold
induction as in Fig. 4. Every compound tested showed
ligand activity on transactivation, nearing saturation at
500 nm while showing only marginal increase at 5 lm.
Among these, JH III, which is one of the native JHs
of Drosophila, was found to be the most effective over
the range of concentrations tested. Of note is that
JH III was conspicuously active in the range of
5–50 nm, whereas the other JHs or JHA showed only
Fig. 4. Ligand specificity of gene activation by MET. The transfec-
tion assay was carried out as described in Fig. 3 with pAcGAL4–
DBD-Met or an empty pAc5.1 ⁄ V5-His A vector as expression con-
structs. S2 cells were incubated with several different compounds
indicated at a concentration of 5 l
M. Activities are shown as fold
induction by dividing the activity obtained with the fusion-expres-
sing construct by that in negative controls using empty vectors
(mean ± SD). The mean value obtained in ethanol controls is taken
as unity.
Fig. 3. MET regulates transcription in a JH-dependent manner. S2
cells were transfected with several different expression constructs
(vector pAc5.1 ⁄ V5-His A, pAcMet, pAcGAL4–DBD, or pAcGAL4–
DBD-Met) together with reporter (pG5luc) and coreporter (pRL-tk)

constructs. After incubation in either the presence or absence of
5 l
M JH III for 24 h, cells were harvested and subjected to dual
luciferase assay. Luciferase activities are shown normalized to that
of the coreporter (mean ± SD).
Fig. 5. Dose–response curves for transactivation by MET with JHs
and JHA. JH I (r), JH II (n), JH III (m), methoprene (x) were inclu-
ded in the culture media in the range from 5 n
M to 5 lM after trans-
fecting S2 cells with the expression (pAcGAL4–DBD-Met or empty
pAc5.1 ⁄ V5-His A), reporter (pG5luc) and coreporter (pRL-tk) con-
structs. Fold induction was calculated as in Fig. 4.
Characterization of Drosophila Met gene product K. Miura et al.
1172 FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS
slight effects. The other native JH of Drosophila, JH-
bisepoxide [34] was not tested. The induction activities
are in the following order: JH III  JH II >
JH I > methoprene. The most effective transcriptional
activation produced by Drosophila MET with its native
JH species further supports the putative role of MET
as a JH-dependent transcription factor. We should
mention here that JHs are highly sticky to glass or
plastic surfaces [35] and would be adsorbed by pipette
tips, test tubes or culture dishes. Thus, the effective
concentrations of these compounds would be lower
than the values indicated in Fig. 5. These data, thus,
indicate that the threshold activity concentration of
JHs is reasonably low in this transient transfection
system.
Localization of MET in S2 cells

In the transfection assays described above, MET was
fused to GAL4–DBD, which has a nuclear localization
sequence. To test the subcellular localization of MET,
we used a fusion to enhanced green fluorescent protein
(EGFP), which does not have a nuclear localization
sequence. S2 cells were transfected with the expression
plasmid pAcMET–EGFP together with the reporter
and coreporter constructs used above. After trans-
fection, cells were incubated for 24 h in the presence
or absence of JH III, then observed by Nomarski
DIC (differential-interference contrast) or fluorescence
microscopy (Fig. 6). In both cases, the fluorescence of
the fusion proteins was seen in the nucleus. In these
experiments the use of cultured cells allows for com-
plete depletion of JH. These observations are consis-
tent with the previous report in vivo [29] and rule out
the ligand-dependent nuclear translocation reported
for the Ahrs of vertebrates [36]. Then, how is JH
transported to the nucleus? A process such as verteb-
rate retinoid transport including cellular retinol-bind-
ing protein [37] may be involved.
Discussion
From its identification as a Drosophila gene responsible
for resistance to morphogenetic and toxic effects of JH
and JHAs, the Met gene product has been implicated
to have an involvement in JH reception. Previous
works on Met do not contradict the hypothesis that
MET may be a component of a JH-dependent tran-
scriptional regulator complex. Direct evidence, how-
ever, for the immediate interaction with JH and

involvement in gene regulation is lacking. To test this
hypothesis, we chose S2 cells as experimental material
because the use of cultured cells would be advanta-
geous for examining ligand-dependent gene regulation
and JH responses in this cell line have been reported
[38–40].
Our principal new contributions are: (a) demonstra-
tion of direct, reversible binding of JH III to MET;
(b) demonstration of its JH-dependent transactivation
potential. The former was enabled by the use of cou-
pled in vitro transcription and translation, as we had
experienced difficulty in obtaining soluble preparations
of full-length bHLH-PAS proteins from mosquitoes
using prokaryotic expression systems (K. Miura,
unpublished data). The binding of JH III by MET
showed high affinity with a nanomolar K
d
value, and
was competed away by an excess of cold JH III.
In the present study, MET was tethered to a promo-
ter by using the GAL4–DBD fusion ⁄ UAS reporter
system. In this heterologous system, the MET fusion
Fig. 6. Subcellular localization of MET in S2 cells. S2 cells were
transfected with pAcMET–EGFP together with the reporter and
coreporter constructs. After transfection for 5 h, cells were incuba-
ted with either 0.5 l
M JH III or ethanol for 24 h. Then, cells were
fixed and observed by Nomarski DIC or fluorescence microscopy.
Horizontal bars represent 10 lm. The results shown are representa-
tive of two independent experiments.

K. Miura et al. Characterization of Drosophila Met gene product
FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS 1173
exhibited specific ligand-dependent activation of a
reporter gene placed downstream of the UAS. The
MET fusion responded only to JH or the JHA metho-
prene while compounds that are structurally related
but hormonally inactive elicited no response. Among
compounds tested, JH III was the most effective lig-
and, even at nanomolar concentrations, which is in
accordance with its nature as one of Drosophila’s
native JHs. The typical range of concentration for
JH In insect haemolymph is 0.3–180 nm [41]. Further,
the maximal JH titre in the Drosophila life cycle is
5–7 pmolÆg
)1
wet weight [12], which would correspond
to 25–35 nm in the haemolymph, assuming that
haemolymph occupies one-fifth of the body weight. In
view of these physiological JH titres, it is thus notable
that in this study JH III was found to be overwhelm-
ingly active in the physiological range of 5–50 nm over
the other JHs or JHA. The ligand-dependent trans-
activation profile exhibited by MET clearly rules out
the possibility that it is simply a JH binder, like cyto-
solic JH binding proteins, and suggests that it might
play a role in JH signaling in vivo.
Recently, Wozniak et al. [42] have reported the con-
formational changes of recombinant Drosophila USP
exposed to several different farnesoid compounds
including natural JHs. They have shown that JH III

and JH I at 100 lm elicit the conformational changes
to a similar degree whereas JH II is the least effective.
Furthermore, by using Drosophila white puparial bio-
assay they have demonstrated that the biochemical dif-
ferences in the three JHs mentioned above parallel the
respective biological activity. For example, in preven-
tion of adult emergence the 50% effective doses
(ED
50
s) for JH I–III are 153, 678 and 143 pmolÆpupa-
rium
)1
, respectively. Another report describes that the
ED
50
of methoprene is 5 pmolÆpuparium
)1
in the same
assay and that Met mutant flies are more resistant to
another JHA, S31183, than the parental fly stock, sug-
gesting the involvement of the Met locus in this resist-
ance [43]. Based on these studies, the order of efficacy
of these compounds in this bioassay seems to be metho-
prene  JH III ‡ JH I > JH II. On the other hand,
the order was JH III  JH II > JH I > methoprene
in our transfection assay. Methoprene is the most
effective in the former and the least effective in the
latter. We do not find this surprising as methoprene is
often highly active over naturally occurring JHs when
applied topically. For example, the early trypsin gene

of Aedes aegypti is upregulated by JH, and low doses
of methoprene, but higher doses of its native JH III
are required to restore its expression in the ligated
abdomens [44]. The higher efficacy of methoprene in
these bioassays may be due to its higher resistance to
enzymatic degradation and possible higher penetration
through the cuticle than natural JHs.
Another point is that JH I has been shown to be
more active than JH II in the white puparial assay [42]
whereas JH II is more active in our transfection assay.
The difference between these two studies is the concen-
trations of JH used. Wozniak et al. [42] used supra-
physiological concentrations in both biochemical and
biological assays whereas we tested JHs or JHA at
much lower range of concentrations. In our assay JH I
and JH II were similarly much less effective than
JH III in the physiological range (5–50 nm) while these
differences were somewhat obscured at higher doses,
although JH II was still more effective than JH I. At
present we do not have data to explain this discrep-
ancy. Possibly, there might be more than one pathway
of JH signaling underlying in the white puparial bio-
assay, one mediated by USP and another by MET.
In the reporter assays, we noted that unliganded
MET repressed the intrinsic activation function pos-
sessed by GAL4–DBD. Although GAL4–DBD is
believed to lack transactivation domains [33], it
showed moderate transactivation potential in Dro-
sophila S2 cells in this study. The nuclear localization
of MET [29] was confirmed by our finding that the

MET–EGFP fusion is concentrated in the nuclei of
transfected S2 cells. In addition, GAL4–DBD has a
nuclear localization sequence. Therefore, it is reason-
able to consider that the GAL4–DBD fusion of MET
sits on the UAS of the reporter construct even in the
absence of ligand, and that the MET moiety is respon-
sible for the observed repression. In the case of verteb-
rate Ahr, a multimeric complex including hsp90
anchors the unliganded Ahr in the cytoplasm, thereby
preventing its transactivation function [36]. Upon lig-
and binding, Ahr translocates to the nucleus and forms
a transcription factor complex with Arnt. In fact, the
C-terminal portion of Ahr fused to GAL4–DBD has
been shown to act as a constitutive activator of gene
regulation [45]. Contrary to this, MET exists in the
nucleus even in the absence of ligand. Upon ligand
binding, it becomes a transcriptional activator. This
resembles the ligand-dependent activation that has
been shown in the activation function-2 of many nuc-
lear hormone receptors [46,47], rather than the case of
the vertebrate Ahr whose activation function is regula-
ted by its subcellular localization.
Two questions arise here as to whether MET func-
tions as a homo- or heterodimer, and as to what DNA
sequences are responsible for the binding of this tran-
scriptional regulator complex. These questions are
related since DNA-binding specificities of bHLH-PAS
proteins are determined by their dimerization properties
Characterization of Drosophila Met gene product K. Miura et al.
1174 FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS

[48]. For example, the dioxin receptor complex
Ahr ⁄ Arnt heterodimer binds to TNGCGTG [25]. Ahr
recognizes the 5¢-half-site TNGC, while Arnt recogni-
zes the 3¢-half-site GTG. Arnt is also capable of
forming a homodimer that recognizes a consensus
palindromic E-box sequence, CACGTG [48]. Dro-
sophila Sim protein forms a heterodimer with Tango (a
Drosophila Arnt-like protein) and binds to ACGTG
core sequence [49]. Thus, DNA binding specificities of
bHLH-PAS dimers are dependent upon the dimer con-
figuration while Arnt or Tango always recognize the
GTG motif. In the present study, we used the GAL4–
DBD fusion of MET in transfection assays. Under
these conditions MET is likely to behave as a homo-
dimer because of its overexpression and because of
dimerization interfaces provided by the GAL4–DBD
moiety. Therefore, the natural dimerization partner
and binding sequence of MET are unknown at present.
Since the bHLH domain of MET shows relatively high
similarity to vertebrate Arnts [19], the use of the con-
sensus sequence CACGTG may be a good starting
point to answer these questions.
Based on the framework by Wilson and coworkers,
our results have further supported the notion that
MET may function as a JH-dependent transcription
factor. In further studies, identification of its target
genes will help elucidate its in vivo function. Molecular
dissection of MET and structural studies may lead to
the development of new biologically active JHA and
new strategies for pest management.

Experimental procedures
JHs, JHA and related compounds
JH I and JH II were obtained from SciTech. JH III was
from Sigma. The JHA, methoprene was a gift from Otsuka
Chemicals Co. Ltd. Squalene, farnesol and geraniol were
from Sigma. Farnesyl acetate was from Aldrich.
cDNA cloning of Met
Total RNA was isolated from S2 cells as described previ-
ously [50]. First-strand cDNA was synthesized by Super-
script reverse transcriptase II (Invitrogen) with oligo dT
primer, and used as a template for RT–PCR. The cDNA
containing a full ORF of Met was amplified by 30 cycles of
PCR using a proofreading polymerase (long and accurate
Taq polymerase, Takara) with the primer pair based on the
published sequence [19]: 5¢-GCCGAATTCCAACATGGC
AGCACCAGAGACGGG-3¢;5¢-GCCTCTAGATCATCG
CAGCGTGCTGGTCAG-3¢. The amplified products were
purified, digested and subcloned into EcoRI and XbaI sites
of pBluescript II (Stratagene), and the identity of the
cDNA clone was confirmed by sequencing.
Binding assay
The DCC assay was carried out as described [51]. Full-
length MET was prepared by a TNT T7 Quick for PCR
DNA Kit (Promega). A cDNA template for the TNT reac-
tion was prepared by PCR using the Met cDNA inserted
downstream of the T7 promoter site of pBluescript II. After
PCR, the cDNA fragments containing the T7 promoter were
purified by a QIAquick PCR purification kit (Qiagen). The
TNT reaction was carried out as follows: 20 lL lysate was
programmed by 400 ng of the cDNA fragment in a total vol-

ume of 25 lL, and the reaction mixture was kept at 30 °C
for 90 min. A reaction in the presence of
35
S-methionine
was performed in parallel, and the lysate was analysed by
SDS ⁄ PAGE and autoradiography to confirm the production
of a polypeptide with the expected size. The DCC assay used
the TNT lysate as a protein source. Each reaction mixture
included 25 lL of the programmed lysate, 74 lL of buffer C
(20 mm Tris ⁄ HCl pH 7.9, 5 mm magnesium acetate, 1 mm
EDTA, 1 mm dithiothreitol) and 1 lL of variable amounts
of
3
H-labeled JH III (specific activity: 17.5 CiÆmmol
)1
,
PerkinElmer) in ethanol in a polyethylene glycol-coated
glass tube. The mixture was incubated at 22 °C for 90 min.
This was followed by the addition of 5% DCC suspension,
gentle mixing for 2 min and centrifugation for 1 min. The
supernatant was collected into a scintillation vial, decolo-
rized overnight with 2 mL 30% H
2
O
2
, and counted by scin-
tillation. Mock-programmed lysates were incubated with the
corresponding amounts of
3
H-labeled JH III and processed

in parallel with the programmed lysates, and the counts
obtained were taken as nonspecific binding. Specific binding
was obtained by subtracting the counts of mock-pro-
grammed lysates from those of corresponding programmed
lysate. The addition of esterase or protease inhibitors in the
binding reaction mixture did not affect binding values (data
not shown). Saturation curves were obtained, and K
d
values
were calculated by the method of Scatchard [52].
Plasmids
The plasmid pAcGAL4–DBD-Met, which expresses a
fusion protein of MET possessing the yeast GAL4–DBD
toward the N terminus, was constructed as follows: a full-
length Met cDNA fragment having overhangs of EcoRV
and XbaI sites was prepared by PCR and subcloned into
pBIND vector (Promega); the cDNA fragment containing
the fused ORF of GAL4–DBD and Met was amplified by
PCR and subcloned into NotI and XbaI sites of pAc5.1 ⁄
V5-His A vector (Invitrogen). The location of the junction
was confirmed by sequencing. A control plasmid pAc-
GAL4–DBD was constructed by inserting the GAL4–DBD
K. Miura et al. Characterization of Drosophila Met gene product
FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS 1175
region of the pBIND vector into the pAc5.1 ⁄ V5-His A
vector. Another control vector pAcMet was prepared by
subcloning the full ORF of Met into the pAc5.1 ⁄ V5-His A
vector. An empty pAc5.1 ⁄ V5-His A vector was used as a
negative control. The reporter plasmid pG5luc, which con-
tains five GAL4 binding sites (UAS) upstream of the firefly

luciferase gene, was from Clontech. The coreporter plasmid
pRL-tk, which expresses Renilla reniformis luciferase,
was from Promega. The plasmid pAcMET–EGFP, which
expresses a fusion protein of MET possessing the EGFP
polypeptide in the C terminus, was constructed by transfer-
ring the fused ORF from pEGFP-N1 vector (Clontech)
to pAc5.1 ⁄ V5-His A vector. The in-frame nature of the
junction was confirmed by sequencing.
Cell culture and transfection
Drosophila S2 cells [53,54] were cultured in Drosophila
Serum-Free Medium (SFM, Invitrogen). The cells were see-
ded at a density of 2.5 · 10
5
cells per well of 24-well plates
one day before transfection. 2 lL of lipofectin (Invitrogen)
per each well was mixed with 25 lL of SFM and incubated
for 40 min at room temperature. One-hundred and fifty
nanograms of DNA (50 ng each of expression, reporter and
coreporter plasmids) per well was mixed with 25 lLof
SFM, and this was combined with the lipofectin ⁄ SFM mix-
ture and incubated for another 15 min. Then, this was
mixed with 200 lL of SFM and overlaid onto S2 cells in
each well. This was followed by 5 h incubation at 27 °C,
and the transfection mixture was replaced by 250 lLof
SFM either containing natural JH (JH I, JH II and JH III),
JHA methoprene, related compounds (squalene, farnesol,
geraniol and farnesyl acetate), or solvent ethanol. The cells
were incubated at 27 °C for another 24 h, lysed and subjec-
ted to the dual luciferase assay (Promega) in a luminometer
(Turner Designs, Model TD-20 ⁄ 20). The reporter activity

was shown as relative luciferase activity by normalizing the
reporter activity to the coreporter activity. Where indicated,
the effect of test compounds were shown as fold induction
by dividing the reporter activity obtained with the pAc-
GAL4–DBD-Met by that in negative controls using empty
pAc vectors. The transfection assay was done at least three
times independently in triplicate, and the reproducibility
was confirmed. The values of relative luciferase activity in
each transfection assay fluctuated a little, but tendency was
always reproducible. In Results, representative data are
shown in the respective figures.
For the observation of the MET–EGFP fusion proteins,
1 · 10
6
of S2 cells were seeded on a 35-mm glass-bottomed
dish (Matsunami Glass, #D110400) 1 day before transfec-
tion. The pAcMET–EGFP was transfected together with
the reporter and coreporter constructs as described above
so as to mimic the transfection conditions of the reporter
assays. After 24 h in either the presence or absence of
0.5 lm JH III, the cells were washed three times in NaCl ⁄ P
i
on the dishes, fixed in 4% (w ⁄ v) paraformaldehyde in
NaCl ⁄ P
i
for 30 min at room temperature, rinsed twice in
NaCl ⁄ P
i
, and observed directly by an inverted microscope
(Nikon, Model TE300) equipped with Nomarski DIC and

fluorescence optics.
Acknowledgements
We thank Drs Takahiro Shiotsuki and Tetsuro Shi-
noda for the help on DCC assay; Dr Gerard R. Wyatt
for reading the manuscript. This work was supported
in part by a grant-in-aid for Scientific Research (C)
to KM (15580049) from the Ministry of Education,
Science, Culture, and Sports of Japan.
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