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Originally published In Press as doi:10.1074/jbc.M104291200 on May 23, 2001
J. Biol. Chem., Vol. 276, Issue 29, 26819-26828, July 20, 2001
Regulation of Ecdysteroid Signaling: Cloning and
Characterization of Ecdysone Oxidase
A NOVEL STEROID OXIDASE FROM THE COTTON LEAFWORM,
SPODOPTERA LITTORALIS*
Hajime
Takeuchi,
Jian-Hua
Chen,
David R.
O'Reilly ,
Philip C.
Turner, and
Huw H.
Rees§
From the Cellular Regulation and Signaling Division, School of
Biological Sciences, University of Liverpool, Life Sciences Bldg.,
Crown Street, Liverpool, L69 7ZB, and Department of
Biology, Sir Alexander Fleming Bldg., Imperial College of Science,
Technology and Medicine, Imperial College Road, South Kensington,
London, SW7 2AZ, United Kingdom
Received for publication, May 11, 2001, and in revised form, May 21, 2001
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ABSTRACT |
One route of inactivation of ecdysteroids in
insects involves ecdysone oxidase-catalyzed conversion into
3-dehydroecdysteroid followed by irreversible reduction by
3-dehydroecdysone 3 -reductase to 3-epiecdysone. We have purified
from Spodoptera littoralis the first ecdysone oxidase and
subjected it to limited amino acid sequencing. A reverse-transcriptase
polymerase chain reaction-based approach has been used to clone the
cDNA (2.8 kilobases) encoding this 65-kDa protein. Northern
blotting showed that the mRNA transcript was expressed in midgut
during the prepupal stage of the last larval instar at a time
corresponding to an ecdysteroid titer peak. Conceptual translation of
the ecdysone oxidase cDNA and data base searching revealed that the
enzyme is an FAD flavoprotein that belongs to the
glucose-methanol-choline oxidoreductase superfamily. Ecdysone oxidase
represents the only oxidase in eukaryotic animals known to catalyze
oxygen-dependent oxidation of steroids; by contrast, oxidation of steroids in vertebrates occurs via
NAD(P)+-linked dehydrogenases. The injection of RH-5992, an
ecdysteroid agonist, induced the transcription of ecdysone oxidase,
suggesting that ecdysone oxidase is an ecdysteroid-responsive gene. The
gene encoding this enzyme, consisting of five exons, has also been isolated. Sequences similar to the binding motifs for Broad-Complex and
FTZ-F1 have been found in the 5'-flanking region. Southern blotting indicated that ecdysone oxidase is encoded by a single-copy gene. We have determined the kinetic characteristics of this novel recombinant ecdysone oxidase produced using a baculovirus expression system.
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INTRODUCTION |
Molting and aspects of reproduction in insects are regulated by
the steroidal molting hormones (ecdysteroids) (1). In immature stages
of insects, the prothoracic glands are the major source of
ecdysteroids, generally ecdysone
(E),1 in most species.
However, in most Lepidoptera examined, the major products of the glands
are 3-dehydroecdysone (3DE), accompanied by varying proportions of
ecdysone (2-6). The observation that interconversion of ecdysone and
3DE by prothoracic glands is not detectable in the cotton leafworm,
Spodoptera littoralis, suggests that 3DE is likely to be an
independent product of pathways of ecdysteroid biosynthesis in the
glands (6). After secretion, 3DE undergoes reduction to ecdysone by
NAD(P)H-linked 3DE 3 -reductase in the hemolymph (3, 4, 6, 7).
Ecdysone then undergoes 20-hydroxylation in certain peripheral tissues,
yielding 20-hydroxyecdysone, which is considered to be the major active
molting hormone in most insect species (8).
At specific stages in development, the ecdysteroid titer exhibits
obligatory, distinct peaks (9). In immature stages, these result from
increased ecdysteroid synthesis in the prothoracic glands, whereas
decreases in titer arise from elevated ecdysteroid inactivation
reactions in conjunction with enhanced excretion. Several
transformations contribute to ecdysteroid inactivation (8), including
the formation of 3-epi (3-hydroxy) ecdysteroids, which are regarded
as hormonally inactive (10-12). Production of 3-epiecdysteroids
occurs in many insect orders but appears to be prominent in
lepidopteran midgut cytosol and entails conversion of ecdysteroid into
3-dehydroecdysteroid followed by NAD(P)H-dependent irreversible reduction to 3-epiecdysteroid (Fig.
1 and Refs. 8 and 12-16). These
reactions occur with both ecdysone and 20-hydroxyecdysone. Ecdysone
oxidase (EC 1.1.3.16) is the enzyme that catalyzes the oxidation of
ecdysteroid and was first demonstrated, characterized, and extensively
purified from the blowfly, Calliphora vicina (17, 18).
However, the 3-dehydroecdysteroid can also undergo
NAD(P)H-dependent 3DE 3-reductase-catalyzed reduction back
to ecdysteroid (for reviews, see Refs. 8 and 19). The significance of
such competitive reactions between ecdysone oxidase and 3DE
3-reductase is uncertain. Furthermore, particularly puzzling is the
occurrence of enzymes for reversible interconversion of ecdysteroid and
their 3-dehydro-derivatives in tissues of several species that are
incapable of producing 3-epiecdysteroids (12, 19).
As part of our studies aimed at elucidating the regulation of
ecdysteroid titer, including the reactions involved in ecdysteroid inactivation in S. littoralis, we have cloned and
characterized the cDNA encoding the 3DE 3-reductase (hemolymph)
(20) and 3DE 3-reductase (Malpighian tubules) (16). Furthermore, we have purified ecdysone oxidase from S. littoralis midgut
plus attached Malpighian tubules, and evidence suggests that the native enzyme consists of a trimer with apparent molecular mass of ~190 kDa
and subunit molecular mass of ~64 kDa (21). Amino acid sequences of
the NH2 terminus as well as of interior tryptic peptides of the purified enzyme have been determined.
We now report the molecular cloning, characterization and heterologous
expression of the cDNA encoding ecdysone oxidase of S. littoralis together with the analysis of the organization of the
corresponding gene and its promoter. Conceptual translation and amino
acid sequence analysis indicates that ecdysone oxidase belongs to the
glucose-methanol-choline (GMC) oxidoreductase superfamily. In
fact, the ecdysone oxidase is novel in being, hitherto, the only
eukaryotic animal steroid-metabolizing oxidase enzyme to be reported
and characterized at the molecular level. By contrast, oxidation of
steroids in vertebrates occurs via NAD(P)+-linked dehydrogenases.
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EXPERIMENTAL PROCEDURES |
Protein Sequence--
Ecdysone oxidase from S. littoralis was purified as described previously (21), subjected to
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel,
electrotransferred to ProBlottTM membrane, and visualized by Coomassie
staining. A single band was observed that was excised and sequenced by
an automated pulsed liquid-phase sequencer (Applied Biosystems 471A),
giving the NH2-terminal amino acid sequence as
XYAVGGXAGAGPAATYVA (where X represents
an unidentified amino acid). To obtain sequences of the interior region
of the enzyme, the purified protein was excised from an SDS-PAGE gel and cleaved with trypsin. The resulting tryptic peptides were purified
by high performance liquid chromatography and sequenced. The best
internal sequence was
ETPYXWXFTTIXXGVT.
cDNA Cloning and Sequencing--
A PCR-based cloning
strategy was used to isolate a cDNA fragment encoding the region
between the two peptide sequences described above. Four degenerate
primers were synthesized. Primers EO3-3 and EO-5 were designed on the
basis of a part of the NH2-terminal amino acid sequence
(EO3-3, 5'-GTN GGN GCI GGI CCI GC, where I represents inosine, and N is
A, T, C, or G; EO-5, 5'-GCN GGI CCN GCI GCN ACN TAY GT, where Y
represents T or C); primers EO-AS3-2 and EO-AS3-3 were designed based
on the internal proteolytic peptide fragment (EO-AS3-2, 5'- ATI GTI GTR
AAI NIC CAI NIR TAI GG, where R represents A or G; EO-AS3-3, 5'-GTI
GTR AAI NIC CAI NIR TAI GGI GT).
Total RNA was extracted using TRIzol (Life Technologies, Ltd.) from
midgut dissected from larvae at 18 h into the last larval instar.
First-strand cDNA was reverse-transcribed from the total RNA using
a first-strand cDNA synthesis kit (Roche Molecular Biochemicals) with random primer p(dN)6 supplied with the kit. cDNA
synthesized with random primer served as the template for PCR in which
the above degenerate primers were used. PCR was carried out as follows: 1 cycle of 94 °C for 3 min and 35 cycles of 94 °C for 1 min,
47 °C for 1 min, 72 °C for 3 min, and 1 cycle of 72 °C for 7 min using EO3-3 and EO-AS3-2 primers. This PCR product was used as a
template for nested PCR, which was carried out as follows: 1 cycle of
94 °C for 3 min and 30 cycles of 94 °C for 1 min, 50 °C for 1 min, 72 °C for 3 min, and 1 cycle of 72 °C for 7 min using EO-5
and EO-AS3-3 primers. The nested PCR yielded a product of ~300
bp.
The purified PCR product was cloned into pGEM®-T Easy
Vector (Promega, Ltd.). Transformants were screened by colony PCR using M13 forward and reverse primers (5'-GTA AAA CGA CGG CCA G and 5'-CAG
GAA ACA GCT ATG AC, respectively), those showing the correct size of
inserts were propagated in LB broth containing 100 µg/ml ampicillin,
and plasmid DNA was purified after a 16-h incubation at 37 °C.
Double-stranded DNA sequencing was performed by the dideoxy termination
method using Sequenase Version 2.0 (usbTM, Amersham Pharmacia
Biotech). The sequences of three independent clones were compared
to detect errors that could have occurred during the reverse
transcription and PCR amplification.
5'-Rapid Amplification of cDNA Ends (5'-RACE)--
5'-RACE
was carried out to obtain the 5'-end of the cDNA. For this,
mRNA from total RNA was isolated using a Dynabeads mRNA purification kit (Dynal Ltd.). A 5'-RACE system, Version 2.0 (Life Technologies, Ltd.) was used to amplify the 5'-terminus of the message for sequencing. Briefly, a gene-specific primer 1 (5'-CTT TCA
AGG TTT GTC TTA A), designed on the basis of the sequence of the PCR
product, was hybridized to the mRNA, and cDNA was synthesized using SUPERSCRIPTTM II reverse transcriptase. The RNA was then degraded
with RNase mix (RNase H and RNase T1), and the cDNA was purified
using a GlassMax spin cartridge supplied with the kit. A poly(dC) tail
was added to the 3' terminus of the purified cDNA using dCTP and
terminal deoxynucleotidyltransferase, and the cDNA region
corresponding to the 5'-end of the mRNA was amplified by two
successive rounds of PCR using additional gene-specific primers 2 and 3 (5'-CCG GTA CAA TGC TCT CCT and 5'-GGT TCG GTC CTG CTT CTA,
respectively), together with the anchor primers supplied by the
manufacturer. The second round PCR yielded a product of ~400 bp,
which was cloned into pGEM®-T Easy Vector, and the
nucleotide sequences of several clones were determined.
Construction and Screening of the Genomic Library--
The
genomic library was constructed using a Lambda FIX®
II/XhoI partial fill-in vector kit (Stratagene). The genomic
DNA was prepared from the fat body and Malpighian tubules of S. littoralis in the last larval instar using a modified phenol
extraction method as described in (22). It was partially digested with
Sau3AI, and the first two nucleotides of the 3' termini were
filled in. The genomic DNA fragments were ligated into the Lambda
FIX® II arms and packaged with Gigapack III Gold packaging extract (Stratagene).
Hybridization to the genomic library was performed for 18 h at
40 °C in 6× SSC (1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate), 5× Denhardt's reagent, 0.5% SDS, 100 µg/ml denatured, fragmented salmon sperm DNA, and a
32P-labeled DNA fragment produced using EO-5 and EO-AS3-3
primers as a probe. Membranes were washed at low stringency (2× SSC,
0.1% SDS, at 40 °C). The positive clones were analyzed by
restriction digestion. The restriction fragments were subcloned into
pBluescript II KS+ (Stratagene) and sequenced.
Determination of Transcription Initiation Site--
The
transcription initiation site of ecdysone oxidase mRNA has been
determined using the solid phase CapFinder approach (23). mRNA from
total RNA was isolated using a Dynabeads mRNA purification kit and
used to synthesize a solid-phase cDNA library with CapFinder Primer
(5'- AAG CAG TGG TAT CAA CGC AGA GTG GCC ATT ATG GCC GGG). The RNA was
then degraded with RNase H, and the immobilized cDNA was purified
by using a magnet. The cDNA region corresponding to the 5'-end of
the mRNA was amplified by two successive rounds of PCR using
gene-specific primers 4 and 5 (5'-GTT CGG TCC TGC TTC TAG CAG CA and
5'-CAC GCT GAA GCG GTT CTC CT, respectively) together with the
5'-primer (5'- AAG CAG TGG TAT C AA CGC AGA GT). The second-round PCR
yielded a product of ~400 bp, which was cloned into
pGEM®-T Easy vector, and the nucleotide sequences of
several clones were determined.
Northern and Southern Blot Analysis--
Total RNA from various
tissues was isolated using TRIzol reagent. mRNA was isolated from
total RNA using a Dynabeads mRNA purification kit. 10 µg of total
RNA or 200 ng of mRNA was fractionated on a formaldehyde/agarose
gel, transferred to Electran® nylon membrane (Merck), and
hybridized with a probe corresponding to the open reading frame of the
ecdysone oxidase cDNA. The probe was radiolabeled with
[-32P]dCTP using a Ready-To-GoTM DNA-labeling kit
(-dCTP) (Amersham Pharmacia Biotech), and loading was normalized
by probing with a mouse 18 S ribosomal RNA probe when using total RNA
or with a S. littoralis muscle actin partial fragment
corresponding to the coding
region2 when using mRNA.
Prehybridization and hybridization were carried out using ULTRAhybTM
(Ambion, Inc.) under the conditions recommended by the manufacturer.
The blots were washed at low stringency (2× SSC and 0.1% SDS at
40 °C), and labeled bands were visualized by autoradiography.
Genomic DNA was prepared as described above. 10-µg aliquots of DNA
were digested with BamHI, EcoRI,
HindIII, or SalI, fractionated on a 1% agarose
gel, transferred to a nylon membrane, and hybridized using radiolabeled
probes corresponding to different regions of ecdysone oxidase cDNA.
Hybridization was carried out in 6× SSC, 5× Denhardt's reagent,
0.5% SDS, and 100 µg/ml denatured, fragmented salmon sperm DNA at
55 °C overnight. The blot was washed at low stringency (2× SSC,
0.1% SDS, at 40 °C), and labeled bands were visualized by autoradiography.
Baculovirus Expression of Ecdysone Oxidase--
Due to
difficulty in amplifying the full length of the coding region, two
fragments (115-1634 and 1589-1914) were amplified using reverse
transcriptase-PCR separately, which were subsequently digested with
EcoRV and ligated. For this PCR step, PLATINUM®
Pfx DNA Polymerase (Life Technologies) was used according to the manufacturer's instructions. This cDNA containing the complete open reading frame of ecdysone oxidase was inserted into pSynXIV VI+X3 vector (24). This construct was cotransfected into
Sf21 cells (~2 × 106 cells) with
vSynVI gal DNA (24). Recombination between viral sequences
flanking ecdysone oxidase cDNA in pSynXIV VI+X3 and
homologous sequences in the viral genome resulted in the replacement of
the -galactosidase in vSynVIgal with the entire
ecdysone oxidase cDNA and a functional polyhedrin gene. The
recombinant viruses were identified by screening based on their
-galactosidase-negative, occlusion-body-positive plaque phenotype.
Their structures were confirmed by restriction endonuclease analysis
followed by Southern blotting. Procedures used for maintenance of
Sf21 cells for propagation of vSynVIgal and for
the construction and characterization of recombinant viruses are
described in O'Reilly et al. (24).
SDS-PAGE--
2 × 106 High FiveTM cells
infected at the multiplicity of infection of 10 with wild type
Autographa californica nucleopolyhedrovirus or with
recombinant baculovirus were homogenized at different times
post-infection in 1 ml of 100 mM Tris buffer (pH 8.0)
containing 0.05% NaN3. The homogenate was centrifuged at
17,000 × g for 10 min, and the supernatant was
recentrifuged at 170,000 × g for 1 h to obtain
the cytosolic fraction. 5 µl of each fraction was added to an equal
volume of SDS sample buffer and boiled for 5 min before loading onto an
8% polyacrylamide gel (22). After electrophoresis, the gel was stained
with Coomassie Brilliant Blue and destained to allow visualization of
the protein.
Enzyme Assay--
Assay of the recombinant ecdysone oxidase
activity was performed in triplicate by modification of the method
described in Chen et al. (21). 2 × 106
High FiveTM cells infected at the multiplicity of infection of 10 with
either wild type or recombinant baculovirus were homogenized at 70 h post-infection in 1 ml of 100 mM Tris buffer (pH 8.0) containing 0.05% NaN3. The homogenate was centrifuged at
17,000 × g for 10 min, and the supernatant was
recentrifuged at 170,000 × g for 1 h to obtain
the cytosol fraction. A 5-µl aliquot was assayed by incubation for 10 min at 40 °C in a 50-µl assay mixture consisting of 0.1 M sodium phosphate buffer (pH 6.6) and 60 µM ecdysone. Assays were quenched with 50 µl of methanol, and proteins were removed by centrifugation. The supernatant was analyzed by reversed-phase HPLC. Protein concentration was determined by the method
of Bradford using a dye binding assay (Bio-Rad) with bovine serum
albumin as a standard. Three independent experiments were performed for
each assay, with all assays carried out in duplicate.
Injection of Insects with 20-Hydroxyecdysone and
RH-5992--
Injections were made via dorsal segments, and injection
sites were sealed with low melting point wax. RH-5992 or
20-hydroxyecdysone was administered to the larvae in methanol (2 µl)
as double injections (1 µg of RH-5992 or 4 µg of 20-hydroxyecdysone
per insect) at 42 and 48 h into the sixth instar.
Methanol-injected larvae served as controls.
Kinetic Studies--
For the kinetic study of ecdysone oxidase,
the reactions were performed as described above, but for 5 min, and
included 1.85 kBq of [23,24-3H2]ecdysone and
various concentrations of unlabeled ecdysone (15).
High Performance Liquid Chromatography (HPLC)--
Ecdysteroids
were analyzed by HPLC through a C18 Nova-Pak cartridge (10 cm × 5 mm; Waters Associates) on a Waters instrument (Waters
Associates) linked to a 440 ultraviolet detector set at 254 nm
and eluted with an isocratic solvent system consisting of
acetonitrile:0.1% (v/v) trifluoroacetic acid in water (22:78 (v/v)) at
1 ml/min (21).
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RESULTS |
Cloning of the Gene Encoding Ecdysone Oxidase--
A PCR-based
cloning strategy, as detailed under "Experimental Procedures,"
allowed us to obtain a cDNA fragment corresponding to the sequence
between nucleotides 139 and 434 in Fig.
2. Gene-specific primers derived from
this sequence were synthesized and used for 5'-RACE to obtain the
5'-end of the cDNA. 5'-RACE produced a cDNA clone of ~390 bp
that contained a putative translation start site at position 115.
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Fig. 2.
Nucleotide and deduced amino acid sequences
of ecdysone oxidase. The cDNA sequence is indicated on the
top line, and the deduced amino acid sequence is on the
second line. Intron positions and lengths are indicated
above the nucleotide sequence. The putative polyadenylation signal is
double-underlined, and the amino acid sequences
obtained from the purified ecdysone oxidase are underlined.
SalI, BamHI, and EcoRV restriction sites are
shown in boxes. Numbers on the right refer to the
last amino acid residue on each line of the respective protein
sequences. Numbers on the left refer to the first residue on
each line of the respective nucleotide sequences.
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A genomic library prepared from mixed tissues of S. littoralis was screened with the initial 295-bp fragment,
resulting in isolation of three clones. Restriction digestion and
Southern blot analysis suggested that the three clones were identical. Gene-specific primers derived from the sequence of the genomic clone
were synthesized and used for reverse transcriptase-PCR and 3'-RACE.
The amplified products were cloned and sequenced. Taken together, all
overlapping cDNAs span a total of 2.8 kb. The polyadenylation
signal (AATAAA) is located at position 2838. As shown in Fig. 2, using
the first ATG as the start codon, the full-length ecdysone oxidase
cDNA encodes a protein of 599 amino acids with a predicted
molecular mass of 65206.1, which is consistent with its apparent
Mr observed in our SDS-PAGE analysis (21). This
conceptually translated protein starts with MCYAVGGC and is consistent
with the NH2-terminal fragment sequence we determined of
XYAVGGX, assuming that the initial methionine has
been cleaved as is commonly observed (25). The first Cys residue may
have been obscured due to the generally high background in the first sequencing cycle coupled with poor sensitivity of detection of this
residue. The protein is mildly acidic with an estimated pI of 5.82.
Genomic Structure of the Ecdysone Oxidase Gene--
Genomic DNA
was prepared from combined fat body and Malpighian tubules and digested
with various restriction enzymes, of which SalI and
BamHI cleavage sites are found in the ecdysone oxidase genomic clone, whereas EcoRI and HindIII sites
are not. The resultant DNA fragments were analyzed by Southern blot
using probes representing different regions of the cDNA. As shown
in Fig. 3, two bands were observed in
BamHI (~2.6 and 2.3 kb) and SalI (~5 and 1 kb)-digested samples when the blot was subjected to hybridization with
a probe, representing the whole coding region of ecdysone oxidase
cDNA. However, when the same blot was re-probed with a cDNA
fragment that corresponded to the NH2-terminal region,
essentially only one band was detected in all samples. Again, a probe
that represents the COOH-terminal region detected only one band in each
case; in the case of EcoRI and HindIII, only a
large band was observed. This analysis is most simply interpreted if
ecdysone oxidase is encoded by a single-copy gene in the S. littoralis genome. A weak band of 2.6 kb in the SalI
digests of Figs. 3, A and C, is most probably due
to a polymorphic SalI site downstream of the gene, since
this genomic DNA was prepared from 10 individuals.
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Fig. 3.
Southern blot analysis of S. littoralis genomic DNA. 10 µg of genomic DNA was
digested with various restriction enzymes as indicated on the
top of each panel. The blot was hybridized with
32P-labeled cDNA corresponding to the entire coding
region (A), a region from the start codon to the
SalI site (115-781, Fig. 1) (B), and a region
from the BamHI site to the stop codon (959-1914, Fig. 1)
(C). DNA marker sizes are indicated on the
left.
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The genomic sequence of ecdysone oxidase showed that the mRNA was
encoded by 5 exons. Exons 3, 4, and 5 contain the coding region (Fig.
4A), and all exon-intron
boundaries followed the "GT-AG rule" for splice donor and acceptor
sequences. A MOTIF (motif.genome.ad.jp) search revealed that the
5'-flanking region of the transcription initiation site contains the
arthropod cap-site motif (26) at  1, a putative TATA box motif at
28, a putative Broad-Complex binding motif (27) at 578, and CF2
binding motifs (28) at 35 and 65 (Fig. 4B). There is a
repeat sequence (TTA)6 at 285 that is likely to be a
microsatellite sequence. However, the 11-bp motif
(CT(G/C)A(G/C)AGTAN(A/T)) is repeated 5 times at 548, 226,
171, 131, and 107, and since it resembles the binding motif for
FTZ-F1, a transcription factor that belongs to the nuclear hormone
receptor superfamily (29, 30), it is very likely that the ecdysone
oxidase in S. littoralis is regulated by a factor of this
type.
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Fig. 4.
Genomic organization of the ecdysone oxidase
gene. A, the five exons are represented by
boxes, with the coding regions hatched.
B, the putative promoter sequence of the ecdysone oxidase
gene. Bold italics indicate transcribed residues (+1 to +20)
The TATA box motif is underlined, and the arthropod cap-site
motif is double-underlined. Predicted transcription factor
binding motifs are indicated by arrows.
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Similarity of the Deduced Amino Acid Sequence of Ecdysone Oxidase
to Proteins of the GMC Oxidoreductase Family--
The deduced amino
acid sequence of the coding region showed similarity to enzymes in the
GMC oxidoreductase family (Fig. 5) such
as Drosophila melanogaster glucose dehydrogenase (49%),
Drosophila pseudoobscura glucose dehydrogenase (49%),
Escherichia coli choline dehydrogenase (47%),
Rhizobium meliloti choline dehydrogenase (45%),
Pseudomonas oleovorans alcohol dehydrogenase (45%),
Aspergillus niger glucose oxidase (38%) and Prunus
serotina (R)-mandelonitrile lyase isoform 1 (40%). The ecdysone oxidase amino acid sequence exhibited features
characteristic of the proteins of the GMC oxidoreductase family (31,
32), such as the four putative FAD binding domains (32) corresponding
to amino acid residues 49-80, 300-319, 548-570, and 578-598,
the flavin attachment loop corresponding to residues 127-144, and the
putative active site (32) at His-538, which are highly conserved in
members of this superfamily (32).
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Fig. 5.
Alignment of the deduced amino acid sequences
of ecdysone oxidase and the most similar data base proteins. The
deduced ecdysone oxidase sequence (EO Spodoptera) was
compared with all sequences in the SWISS-PROT data base using the BLAST
program. Only the amino acid sequences of the most similar proteins
present in the GMC oxidoreductase family are shown in the alignment.
Gaps introduced to optimize alignment are indicated by
hyphens. Identical amino acids between ecdysone oxidase and
at least one other sequence are indicated in black boxes.
Numbers on the right refer to the last amino acid residue on
each line of the respective protein sequences. The accession number and
the percentage similarity to the ecdysone oxidase sequence (EO
Spodoptera) of each protein are as follows: D. melanogaster glucose dehydrogenase (GDH), P18173, 49%;
D. pseudoobscura glucose dehydrogenase (GDH),
P18172, 49%; E. coli choline dehydrogenase
(CHD), P17444, 47%; R. meliloti choline
dehydrogenase (CHD), P54223, 45%; P. oleovorans
alcohol dehydrogenase (ADH), Q00593, 45%; A. niger glucose oxidase (GOD), P13006, 38%; P. serotina (R)-mandelonitrile lyase isoform 1 (LYA), P52706, 40%. The alignment was constructed by use of
the CLUSTALW program.
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Tissue Distribution and Developmental Expression of Ecdysone
Oxidase--
As demonstrated in Fig. 6,
a cDNA probe representing the protein-coding region of ecdysone
oxidase detected a transcript (2.8 kb) in midgut prepared from the
prepupal stage of the last larval instar. No detectable expression at
this stage of development was found in fat body, Malpighian tubules,
hemocytes, and epidermis.
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Fig. 6.
Northern blot analysis of the tissue
distribution of ecdysone oxidase. A 10-µg aliquot of total RNA
from various tissues during the prepupal stage of the last larval
instar was used. The blot was hybridized with 32P-labeled
ecdysone oxidase cDNA probe, which corresponded to the whole
protein coding region. The same blot was stripped and reprobed with 18 S rRNA probe.
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The quantitative profile of the specific activity of ecdysone oxidase
during the last instar is shown in Fig.
7A. The ecdysone oxidase
activity was detected from an early stage of the last larval instar,
and it reaches a peak of 75 nmol/h/mg at 103 h, just after a time
when the ecdysteroid titer (data are from Chen et al. Ref.
6) in hemolymph reaches a peak. The activity quickly decreased with the
onset of pupation and increased again during the pupal stage.
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Fig. 7.
The developmental profile of ecdysone oxidase
in midgut during the sixth instar. A, profile of the
enzymatic activity. Boxes on the abscissa refer
to the light and dark phases, and insects were synchronized at the
fifth/sixth instar molt, occurring over a scotophase. Each point
represents the mean of three separate investigations, with each assay
carried out in duplicate; bars represent the S.E.  ,
enzymatic activity;  , ecdysteroid titer in hemolymphs (ecdysone
equivalents were measured by radioimmunoassay in a different batch of
insects; data are from Chen et al. (6)). G is gut
purge. W is wandering. B, Northern blot analysis.
200 ng of poly(A)+ RNA from midgut at various time points
within the sixth larval instar, as indicated, was used. The blots were
hybridized with a 32P-labeled ecdysone oxidase cDNA
probe corresponding to the open reading frame.
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Northern blot analysis of mRNA isolated from midgut at different
developmental stages of the last larval instar (Fig. 7B) revealed that the ecdysone oxidase mRNA is mainly expressed in the
prepupal stage of the instar and in the early pupal stage. The mRNA
started to be detectable at 20 h into the last larval instar,
although its expression level during the feeding stage was very low. It
started to increase in intensity from 66 h, reached a peak at
96 h, quickly decreased just before pupation, and rose again after
pupation. The developmental profile of ecdysone oxidase mRNA
expression slightly preceded that of the enzyme activity. Attempts to
normalize the Northern blot have proven problematic because of
developmental changes in the transcript levels of the probes used for normalization.
Induction of the Ecdysone Oxidase Transcript by
20-Hydroxyecdysone and RH-5992--
To examine possible induction of
ecdysone oxidase mRNA transcription, we injected larvae twice at 42 and 48 h into the last larval instar with 20-hydroxyecdysone or
RH-5992, i.e. before the natural increase in enzymatic
activity. Total RNA was isolated from the midgut of the larvae 4 or
18 h after the final injection and analyzed by Northern blotting.
The mRNA encoding ecdysone oxidase was strongly induced by RH-5992
after the final injection (Fig. 8).
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Fig. 8.
Effect of 20-hydroxyecdysone and agonist on
the induction of ecdysone oxidase mRNA. Last instar larvae
were injected with methanol, 2 µg of 20-hydroxyecdysone
(20E), or 1 µg of RH-5992 at 42 h and 48 h into
the sixth instar and dissected at the times indicated. 200 ng of
poly(A)+ RNA was analyzed by Northern blotting using
32P-labeled ecdysone oxidase (coding region) or actin
cDNA as probes.
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|
Expression of Ecdysone Oxidase in High FiveTM
Cells--
Monolayers of High FiveTM cells were infected with wild
type or with recombinant baculovirus containing the coding region of ecdysone oxidase, and the cells were collected at intervals between 24 and 70 h. A polypeptide of ~65 kDa, as determined by SDS-PAGE, increased in intensity with time post-infection in samples infected with the recombinant baculovirus but not in samples infected with the
wild type virus (Fig. 9).
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Fig. 9.
SDS-PAGE analysis of protein synthesis in
High FiveTM cells infected with recombinant or wild-type
baculovirus. Infected cells were lysed and collected at 24, 41, 48, and 70 h post-infection. Each extract was analyzed by SDS-PAGE
and visualized by Coomassie Brilliant Blue (see "Experimental
Procedures"). The positions of the protein markers are shown on the
left. The position of ecdysone oxidase is shown on the
right.
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The recombinant virus-infected cell lysate was enzymatically functional
in oxidizing ecdysone to 3-dehydroecdysone (Fig.
10). As shown in the reversed-phase
HPLC UV chromatograms, there was appreciable conversion of ecdysone
into 3-dehydroecdysone from ecdysone oxidase recombinant virus-infected
cell lysate, with no detectable conversion in the case of wild-type
virus-infected cell lysate or uninfected High FiveTM cell lysate alone
(data not shown). The product of the enzyme reaction was purified
by HPLC and analyzed by liquid secondary ion mass spectrometry using
glycerol matrix and a Cs+ primary ion beam on a VG Quattro
triple quadrupole mass spectrometer (VG Biotech, Altrincham, UK). In
the negative ion spectrum, the most prominent ion was at
m/z 461 [M-H], consistent with an
Mr of 462 as expected for 3-dehydroecdysone.
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Fig. 10.
Enzymatic activity of recombinant ecdysone
oxidase. Cytosol fractions of High FiveTM cells infected with
wild-type or recombinant baculovirus were prepared and incubated with
ecdysone, and the products were analyzed by reversed-phase HPLC (see
"Experimental Procedures"). The positions of elution of authentic
ecdysone (E), 3-epiecdysone (E'), and
3-dehydroecdysone (3DE) are shown. abs.,
absorbance.
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Kinetic Parameters--
The kinetic parameters
Km and Vmax of recombinant
ecdysone oxidase were investigated. The effect of substrate
concentration on the activity of ecdysone oxidase is shown in Fig.
11. A characteristic hyperbolic curve
was observed, typical of an enzyme obeying Michaelis-Menten kinetics.
The Km and Vmax values
determined from the hyperbola (5.10 µM and 0.036 nmol/min/µg of protein) and Hanes plot (5.80 µM and
0.038 nmol/min/µg of protein) were broadly in agreement.
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Fig. 11.
Effect of various concentrations of ecdysone
on the velocity of ecdysone oxidase. Recombinant ecdysone oxidase
(cytosolic fraction) was incubated under standard enzyme activity
assay conditions (see "Experimental Procedures"). Each assay was
performed in duplicate, and the data presented are the means for three
independent experiments. The inset shows the Hanes plot of
ecdysone oxidase activity.
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DISCUSSION |
3-Epimerization of ecdysteroids is one of the inactivation
pathways of these hormones, and ecdysone oxidase converts ecdysteroids into 3-dehydroecdysteroids as intermediates in the formation of 3-epiecdysteroids (Fig. 1). Using a reverse transcription-PCR-based cloning strategy employing degenerate primers designed on the basis of
the partial amino acid sequences of the purified S. littoralis ecdysone oxidase together with 5'- and 3'-RACE, the
complete cDNA encoding the enzyme was isolated and sequenced. The
predicted amino acid sequence of the cDNA contained our
experimentally determined NH2-terminal and internal
proteolytic peptide sequences of the enzyme. Furthermore, when the
cDNA encoding ecdysone oxidase was expressed using a baculovirus
expression system, a polypeptide band of ~65 kDa was observed on
SDS-PAGE that increased in intensity with the time of culture (Fig. 9).
The lysate from recombinant baculovirus-infected cells showed ecdysone
oxidase enzymatic activity. Collectively, these results indicate that
the cloned cDNA encodes ecdysone oxidase.
Southern blot analysis indicates that ecdysone oxidase is probably a
unique gene in the haploid genome of S. littoralis,
consisting of five exons. Exons 3, 4, and 5 contain the coding region
(Fig. 4A). Exon-intron boundaries follow the "GT-AG
rule" for the splice donor and acceptor sequences. There are motifs
corresponding to an arthropod cap-site at 1, the putative TATA box at
28, and the CF2 binding motifs at 35 and 65 in the 5'-flanking
region (Fig. 4B).
Data base searching revealed that ecdysone oxidase belongs to the GMC
oxidoreductase superfamily (31, 32). It is clear that the predicted
amino acid sequence of this enzyme has significant similarity to other
enzymes in this family and that, in particular, four putative FAD
binding regions (amino acid residues 49-80, 300-319, 548-570, and
578-598; Ref. 32), the flavin attachment loop (residues 127-144), and
His-538, essential for catalytic function, are conserved (32). These
data indicate that ecdysone oxidase in S. littoralis is an
FAD flavoprotein. It is noteworthy that ecdysone oxidase has low
sequence similarity (27%) to another FAD-dependent enzyme
catalyzing 3-oxidation of a sterol, cholesterol oxidase from
Brevibacterium sterolicum (33). However, in the case of this
substrate, the conformation of the sterol ring structure is quite
different from that in ecdysteroids owing to the A/B cis
ring junction in the latter. Furthermore, the significance of the
observation that the primary sequence alignment shows that ecdysone
oxidase has highest similarity to glucose dehydrogenase from
Drosophila is unclear. Although ecdysone oxidase activity has been reported in Drosophila (34), no sequence has been
annotated as a putative ecdysone oxidase. There is some significant
similarity in gene structure between the ecdysone oxidase in S. littoralis and the two Drosophila glucose dehydrogenase
genes. All three have a large intron close to the transcription start
site and upstream of the start codon. The last, relatively small intron largely separates the first FAD binding domain from the remainder of
the coding region, and consequently, the last exon is large. However
the S. littoralis ecdysone oxidase gene has an additional intron compared with these glucose dehydrogenase genes. The P. serotina (R)-mandelonitrile lyase gene has a different structure.
Ecdysone oxidase activity has been demonstrated in midgut, fat body,
hemolymph, and integument of larvae in various insect species, although
the predominant activity appears to be in midgut during the larval
stage (11, 35, 36). Our Northern blot analysis shows that the mRNA
for ecdysone oxidase was highly expressed in the midgut but
undetectable in other tissues examined at the prepupal stage in
S. littoralis under our conditions (Fig. 6). These data
indicate that midgut is the main tissue that can produce ecdysone
oxidase during the prepupal stage.
The developmental profile of the enzymatic activity (Fig. 7) revealed
that the ecdysone oxidase is predominantly expressed during the
prepupal stage of the last larval instar. Similarly, Northern blot
analysis revealed that the expression pattern of the mRNA
corresponds closely to the enzyme activity profile. This result
suggests that ecdysone oxidase is primarily regulated at the
transcriptional level. The fact that high expression of ecdysone oxidase occurs in the late stage of the last larval instar, when the
active molting hormone titer is highest (6), suggests that the enzyme
may play a role in deactivation of endogenous ecdysteroids. In
Manduca sexta, ecdysone oxidase activity also increases at a
similar developmental stage as observed in S. littoralis
(36). Also, the developmental profile showed that ecdysone oxidase is highly expressed in the early pupal stage. In many insects, high ecdysone oxidase activity has been detected in pupae (35), but the
significance of this enzyme in the pupal stage is unclear.
In M. sexta it has been shown that ecdysone oxidase activity
can be induced by the ecdysteroid agonist, RH-5849, but not by 20-hydroxyecdysone (37). Similarly, in the current work, mRNA for
ecdysone oxidase is induced by the ecdysteroid agonist, RH-5992, with
no clear induction by 20-hydroxyecdysone (Fig. 8). Furthermore, in vivo expression of this enzyme seems to developmentally
follow to some extent the ecdysteroid titer determined previously in hemolymph of a different batch of insects (Fig. 7). These data suggest
that the gene encoding ecdysone oxidase is ecdysteroid-responsive. Although there is no obvious match to the ecdysone receptor binding motif (38, 39) in the 5'-flanking region of this gene (Fig. 4B), there is an apparent Broad-Complex binding motif (27), which is an ecdysone-responsive element that is known to be required for induction of late genes. Interestingly, there are five copies of a
motif in this promoter region, similar to the FTZ-F1 binding motif
(30). FTZ-F1 is a member of the nuclear hormone receptor superfamily
and is expressed in the prepupal stage (40, 41); it is induced by
ecdysteroids and functions as a regulatory factor for the late genes
(30, 42). Therefore, it is quite possible that ecdysone oxidase is
regulated by FTZ-F1 or a similar factor.
Ecdysteroid 26-hydroxylase, another ecdysteroid inactivation enzyme, is
also induced by ecdysteroids in S. littoralis (43). It is
possible that both these inactivation systems may have similar regulatory mechanisms. Both enzymes catalyze the first steps in ecdysteroid inactivation pathways and might be expected to be subject
to regulation. By contrast, 3DE 3-reductase and ecdysone 20-monooxygenase, which are responsible for the activation of ecdysteroids, are not induced by ecdysteroids (37,
43).3
We characterized the kinetics of ecdysone oxidase using the recombinant
protein. The results revealed that the Km for
ecdysone of this recombinant enzyme (5 µM) from S. littoralis was similar to that (20 µM) previously
reported for the partially purified enzyme (15) and was of a similar
order of magnitude as those from other species (the blowfly, C. vicina, 42 µM (17), 98 µM (18), and
M. sexta, 13.3 µM (44)).
We previously reported (20) the deduced amino acid sequence of 3DE
3-reductase from S. littoralis, which catalyzes
NAD(P)H-dependent reduction of 3DE to yield ecdysone.
Although this enzyme catalyzes the reverse conversion to that of
ecdysone oxidase, it belongs to the different, aldo-keto reductase
superfamily, and there is no significant sequence similarity between
them. From this it would seem that these two distinct S. littoralis enzymes have evolved independently. A different
situation exists in vertebrates where nicotinamide nucleotide
cofactor-dependent 3-hydroxysteroid dehydrogenases
generally catalyze reversible oxidation-reduction reactions at C-3 of
steroids. In fact, oxygen-dependent oxidase-catalyzed oxidation of steroids in vertebrates has not been reported previously, thus making ecdysone oxidase novel among eukaryotic animals. Taken together, these facts suggest that major parts of the system of steroid
conversion in insects are evolutionarily distinct from those of vertebrates.
| |
FOOTNOTES |
*
This work was supported by a grant from the Biotechnology
and Biological Sciences Research Council.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AY035784 and AY035785.
§
To whom correspondence should be addressed: Cellular Regulation and
Signaling Division, School of Biological Sciences, University of
Liverpool, Life Sciences Bldg., Crown St., Liverpool, L69 7ZB, UK.
Tel.: 44 151 794 4352; Fax: 44 151 794 4349; E-mail:
reeshh@liv.ac.uk.
Published, JBC Papers in Press, May 23, 2001, DOI 10.1074/jbc.M104291200
2
H. Takeuchi, J.-H. Chen, D. R. O'Reilly,
P. C. Turner, and H. H. Rees, unpublished data.
3
J.-H. Chen, unpublished data.
| |
ABBREVIATIONS |
The abbreviations used are:
E, ecdysone;
3DE, 3-dehydroecdysone;
GMC, glucose-methanol-choline;
PAGE, polyacrylamide
gel electrophoresis;
PCR, polymerase chain reaction;
RACE, rapid
amplification of cDNA ends;
bp, base pair(s);
HPLC, high
performance liquid chromatography;
kb, kilobases.
| |
REFERENCES |
| 1.
|
Koolman, J.
(ed)
(1989)
Ecdysone
, Georg Thieme-Verlag, Stuttgart, Germany
|
| 2.
|
Warren, J. T.,
Sakurai, S.,
Rountree, D. B.,
Gilbert, L. I.,
Lee, S. S.,
and Nakanishi, K.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
958-962
|
| 3.
|
Sakurai, S.,
Warren, J.,
and Gilbert, L. I.
(1989)
Arch. Insect Biochem. Physiol.
10,
179-197
|
| 4.
|
Kiriishi, S.,
Rountree, D. B.,
Sakurai, S.,
and Gilbert, L. I.
(1990)
Experientia (Basel)
46,
57-64
|
| 5.
|
Blais, C.,
and Lafont, R.
(1991)
C. R. Acad. Sci. (Paris) Ser. III
313,
359-364
|
| 6.
|
Chen, J.-H.,
Webb, T. J.,
Powls, R.,
and Rees, H. H.
(1996)
Eur. J. Biochem.
242,
394-401
|
| 7.
|
Watson, R. D.,
Spaziani, E.,
and Bollenbacher, W. E.
(1989)
in
Ecdysone
(Koolman, J., ed)
, pp. 188-196, Georg Thieme-Verlag, Stuttgart, Germany
|
| 8.
|
Rees, H. H.
(1995)
Eur. J. Entomol.
92,
9-39
|
| 9.
|
Steel, C. G. H.,
and Vafopoulou, X.
(1989)
in
Ecdysone
(Koolman, J., ed)
, pp. 221-231, Georg Thieme-Verlag, Stuttgart, Germany
|
| 10.
|
Nigg, H. N.,
Svoboda, J. A.,
Thompson, M. J.,
Kaplanis, J. N.,
Dutky, S. R.,
and Robbins, W. E.
(1974)
Lipids
9,
971-974
|
| 11.
|
Blais, C.,
and Lafont, R.
(1984)
Hoppe-Seyler's Z. Physiol. Chem.
365,
809-817
|
| 12.
|
Milner, N. P.,
and Rees, H. H.
(1985)
Biochem. J.
231,
369-374
|
| 13.
|
Weirich, G. F.
(1989)
in
Ecdysone
(Koolman, J., ed)
, pp. 174-180, Georg Thieme-Verlag, Stuttgart, Germany
|
| 14.
|
Webb, T. J.,
Powls, R.,
and Rees, H. H.
(1995)
Biochem. J.
312,
561-568
|
| 15.
|
Webb, T. J.,
Powls, R.,
and Rees, H. H.
(1996)
Insect Biochem. Mol. Biol.
26,
809-816
|
| 16.
|
Takeuchi, H.,
Chen, J.-H.,
O'Reilly, D. R.,
Rees, H. H.,
and Turner, P. C.
(2000)
Biochem. J.
349,
239-245
|
| 17.
|
Koolman, J.,
and Karlson, P.
(1975)
Hoppe-Seyler's Z. Physiol. Chem.
356,
1131-1138
|
| 18.
|
Koolman, J.,
and Karlson, P.
(1978)
Eur. J. Biochem.
89,
453-460
|
| 19.
|
Lafont, R.,
and Koolman, J.
(1984)
in
Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones
(Hoffmann, J.
, and Porchet, M., eds)
, pp. 196-226, Springer-Verlag, Berlin
|
| 20.
|
Chen, J.-H.,
Turner, P. C.,
and Rees, H. H.
(1999)
J. Biol. Chem.
274,
10551-10556
|
| 21.
|
Chen, J.-H.,
Powls, R.,
Rees, H. H.,
and Wilkinson, M. C.
(1999)
Insect Biochem. Mol. Biol.
29,
899-908
|
| 22.
|
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual
, 2nd Ed.
, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
|
| 23.
|
Schramm, G.,
Bruchhaus, I.,
and Roeder, T.
(2000)
Nucleic Acids Res.
28,
e96
|
| 24.
|
O'Reilly, D. R.,
Miller, L. K.,
and Luckow, V. A.
(1994)
Baculovirus Expression Vectors: A Laboratory Manual
, Oxford University Press, New York
|
| 25.
|
Varshavsky, A.
(1992)
Cell
69,
725-735
|
| 26.
|
Cherbas, L.,
and Cherbas, P.
(1993)
Insect Biochem. Mol. Biol.
23,
81-90
|
| 27.
|
von Kalm, L.,
Crossgrove, K.,
Von Seggern, D.,
Guild, G. M.,
and Beckendorf, S. K.
(1994)
EMBO J.
13,
3505-3516
|
| 28.
|
Gogos, J. A.,
Hsu, T.,
Bolton, J.,
and Kafatos, F. C.
(1992)
Science
257,
1951-1954
|
| 29.
|
Lavorgna, G.,
Ueda, H.,
Clos, J.,
and Wu, C.
(1991)
Science
252,
848-851
|
| 30.
|
Ayer, S.,
and Benyajati, C.
(1992)
Mol. Cell. Biol.
12,
661-673
|
| 31.
|
Cavener, D. R.
(1992)
J. Mol. Biol.
223,
811-814
|
| 32.
|
Kiess, M.,
Hecht, H.-J.,
and Kalisz, H. M.
(1998)
Eur. J. Biochem.
252,
90-99
|
| 33.
|
Ohta, T.,
Fujishiro, K.,
Yamaguchi, K.,
Tamura, Y.,
Aisaka, K.,
Uwajima, T.,
and Hasegawa, M.
(1991)
Gene
103,
93-96
|
| 34.
|
Sommé-Martin, G.,
Colardeau, J.,
and Lafont, R.
(1988)
Insect Biochem.
18,
729-734
|
| 35.
|
Koolman, J.
(1978)
Hoppe-Seyler's Z. Physiol. Chem.
359,
1315-1321
|
| 36.
|
Weirich, G. F.,
Feldlaufer, M. F.,
and Svoboda, J. A.
(1993)
Arch. Insect Biochem. Physiol.
23,
199-211
|
| 37.
|
Williams, D. R.,
Chen, J.-H.,
Fisher, M. J.,
and Rees, H. H.
(1997)
J. Biol. Chem.
272,
8427-8432
|
| 38.
|
Cherbas, L.,
Lee, K.,
and Cherbas, P.
(1991)
Genes Dev.
5,
120-131
|
| 39.
|
Luo, Y.,
Amin, J.,
and Voellmy, R.
(1991)
Mol. Cell. Biol.
11,
3660-3675
|
| 40.
|
Woodard, C. T.,
Baehrecke, E. H.,
and Thummel, C. S.
(1994)
Cell
79,
607-615
|
| 41.
|
Sun, G.-C.,
Hirose, S.,
and Ueda, H.
(1994)
Dev. Biol.
162,
426-437
|
| 42.
|
Murata, T.,
Kageyama, Y.,
Hirose, S.,
and Ueda, H.
(1996)
Mol. Cell. Biol.
16,
6509-6515
|
| 43.
|
Chen, J.-H.,
Kabbouh, M.,
Fisher, M. J.,
and Rees, H. H.
(1994)
Biochem. J.
301,
89-95
|
| 44.
|
Weirich, G. F.,
Thompson, M. J.,
and Sovoboda, J. A.
(1989)
Arch. Insect Biochem. Physiol.
12,
201-218
|
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ABSTRACT
INTRODUCTION
