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The expression patterns of a eukaryotic initiation factor 3 subunit H in the silk glands in Bombyx mori.

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A r t i c l e
THE EXPRESSION PATTERNS OF A
EUKARYOTIC INITIATION FACTOR
3 SUBUNIT H IN THE SILK GLANDS
IN Bombyx mori
Jia-Lin Wang, Li-fu Wang, Jin-Xing Wang, and
Xiao-Fan Zhao
School of Life Sciences, Shandong University, Jinan 250100,
Shandong, China
Eukaryotic initiation factor 3 subunit H has been characterized in many
organisms, and it has been found to play many roles including help
regulate translation initiation. In this work, we studied the tissue
distribution and expression profiles of Bombyx mori (B. mori) eIF3
subunit H (BmeIF3h). BmeIF3h was prominently expressed in silk
glands, with anterior silk glands (ASGs), middle silk glands (MSGs),
and posterior silk glands (PSGs) all expressing BmeIF3h. The expression
levels of BmeIF3h in MSGs and PSGs were higher than that in ASGs
during 0 d and 2 d of the 5th instar larvae. The expression levels of
BmeIF3h in MSGs and PSGs were up-regulated once the silk glands
began to synthesize silk protein during the feeding stage of the 4th instar
larvae. Immunohistochemistry showed that BmeIF3h was distributed in
the cytoplasm of MSGs cells and in both the nucleus and the cytoplasm of
PSGs cells. These data suggest that BmeIF3h had different action
behaviors in the MSGs and PSGs related to the production of the silk
C 2010 Wiley
glue proteins and silk fibre proteins, respectively. Periodicals, Inc.
Keywords: silk glands; BmeIF3h; expression profiles; hormone regulation
Grant sponsor: National High Technology Research and Development Program of China; Grant number:
2006AA10A119; Grant sponsor: National Natural Science Foundation of China; Grant number: 30710103901.
Correspondence to: Xiao-Fan Zhao, School of Life Sciences, Shandong University, Jinan 250100, Shandong,
China. E-mail: xfzhao@sdu.edu.cn
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 75, No. 1, 1–12 (2010)
Published online in Wiley Online Library (wileyonlinelibrary.com).
& 2010 Wiley Periodicals, Inc. DOI: 10.1002/arch.20369
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Archives of Insect Biochemistry and Physiology, September 2010
INTRODUCTION
The eukaryotic initiation of protein synthesis is a multistep process requiring the
participation of a large number of translation initiation factors (eIFs). Among these
translation initiation factors, eukaryotic initiation factor 3 (eIF3), the largest multisubunit complex, plays an essential role in the rate-limiting initiation phase of
translation (Hinnebusch, 2006). At the beginning of the translation, eIF3 promotes the
formation of 43S pre-initiation complex containing the Met-tRNAieIF2GTP ternary
complex, eIF1, eIF1A, eIF3, and the 40S ribosomal subunit (Chaudhuri et al., 1999).
By interacting with eIF4G, the largest member of the eIF4F cap-binding complex,
eIF3, then promotes the binding of 50 -m7G-capped mRNA (Lamphear et al., 1995;
Korneeva et al., 2000), followed by scanning along the mRNA for AUG translational
initiation codon and the formation of the 48S pre-initiation complex. Finally, with the
release of eIFs, the 60S ribosomal subunit joins the 48S complex to form the 80S
ribosome for elongation (Pestova et al., 2000).
Thirteen putative subunits known as eIF3a to eIF3m have been characterized in
mammalian cells (Browning et al., 2001). In contrast, eIF3 from yeast contains only
five core subunits and several non-core subunits (Browning et al., 2001; Hinnebusch,
2006). Core subunits, which are conserved from yeast to human, tend to be
indispensable for cell growth, while non-core subunits, which are less conserved,
remain poorly understood. Studies regarding the role of eIF3 subunits in translational
initiation were mostly performed in yeast, because of the complexity of mammalian
eIF3.
eIF3h (also called eIF3p40), with an approximate molecular mass of 40 kDa, is one
of subunits of eIF3. Ray et al. (2008) demonstrated that the deletion of eIF3h did not
influence various parameters of protein synthesis in fission yeast, but affected spore
formation, and human eIF3h can functionally substitute fission yeast eIF3h in vivo
complementation. However, many human cancer cells contain high levels of eIF3h,
and over-expression of eIF3h in stably transfected NIH-3T3 cells results in a number
of oncogenic properties (Zhang et al., 2007). Furthermore, Zhang et al. (2008)
demonstrated that high levels of eIF3h directly stimulate initiation and global protein
synthesis rates, leading to the establishment and maintenance of the malignant state in
cells. Data from Arabidopsis thaliana indicated that eIF3h was not essential for general
protein translation, but it may control the translation of specific mRNAs (Kim et al.,
2004).
The homologue of eIF3h in B. mori (BmeIF3h), EN10, has been isolated from
anterior silk glands as one of the ecdysteroid-inducible genes in programmed cell
death (PCD) during pupal metamorphosis (Tsuzuki et al., 2001). However, there was
no further study on the function of BmeIF3h in the silk glands.
The silk glands of B. mori, are divided into three morphologically and functionally
distinct compartments that are the sites of silk protein synthesis: the anterior silk gland
(ASG), the middle silk gland (MSG), and the posterior silk gland (PSG). The PSGs and
MSGs produce the silk fibre proteins and silk glue proteins, respectively, while the
ASGs serve as ducts to transport the silk protein (Dhawan and Gopinathan, 2003).
After the embryonic stage, silk gland cells perform endoreduplication and operate for
17 to 19 rounds of DNA replication in MSG and PSG nuclei during larval stages,
resulting in a large number of gene copy numbers per unit cell (Perdrix-Gillot, 1979).
This shows that the synthesis of silk protein is proportional to the gene dosage during
the last instar (Dhawan and Gopinathan, 2003).
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Eukaryotic Initiation Factor 3 Subunit H
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To further understand the function of BmeIF3h in the silkworm, we examined the
distribution of BmeIF3h in various tissues. We also investigated the expression profiles
of BmeIF3h in the time course of silk glands in development. Furthermore, we
examined the hormonal regulation on the expression of BmeIF3h. Using immunohistochemical techniques, the subcellular distribution of BmeIF3h proteins in MSGs and
PSGs was studied as well. These results suggest that BmeIF3h is associated with silk
protein synthesis in MSGs and PSGs.
MATERIALS AND METHODS
Insects
The silkworm strain Qingsong Haoyue was used in our experiments. The larvae
were reared on an artificial diet (Nihon Nosanko, Yokohama, Japan) at 251C on a 12-hlight/12-h-dark photoperiod.
Amplification of the ORF of BmeIF3h
Total RNA was isolated from the silk glands of several 5th instar larvae 2 d post-ecdysis
using Unizol reagent (Biostar, Shanghai, China). Five micrograms of total RNA was
used to synthesize first-strand cDNA with MMLV reverse transcriptase (Clontech,
Mountain View, CA) according to the manufacturer’s instructions. We searched the
NCBI database to obtain the cDNA of BmeIF3h (GenBank accession no.
NP_001036848). Two specific primers, eIF3h pET F (50 -tactcagaattcatggcgagccgtgctggttcagc-30 ) and eIF3h pET R (50 -tactcactcgagctagttgttttgtttcgcttctttcg-30 )
were designed from the start and stop sites of the BmeIF3h cDNA sequence,
respectively, and was used to amplify the complete ORF of BmeIF3h. The amplified
product was cloned into a pMD-18-T vector (TaKaRa, Shiga, Japan) and was
subsequently sequenced.
RT-PCR
The total RNA of various tissues (epidermis, midguts, fat bodies, and silk glands) was
extracted from several 5th instar larvae 0 d post-ecdysis. RNA (5 mg) was used to
reverse transcribe first cDNA (First Strand cDNA Synthesis Kit, Sangon, Shanghai,
China), which was used as a template in the PCR reactions with gene-specific primers
eIF3h pET F and eIF3h pET R. The PCR cycles were as follows: 1 cycle (941C, 2 min),
28 cycles (941C, 30 s; 601C, 45 s; 721C, 1 min), and the last cycle (721C, 10 min).
A b-actin gene fragment was also amplified as control with primers Actin 3F
(50 -cgacgtggacatccgtaagg-30 ) and Actin 3R (50 -cttcctgtgtacaatggaggg-30 ).
Expression of rBmeIF3h and Preparation of Anti-BmeIF3h Serum
The ORF of BmeIF3h cDNA was subcloned into the EcoRI and XhoI sites of expression
vector pET-30a. Subsequently, the constructed plasmids were transformed into the
E. coli BL21 (DE3) expression host. Luria-Bertani broth (200 ml) was inoculated with
2 ml of overnight culture and was shaken at a temperature of 371C. When the OD600
reached 0.8–1.0, isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to induce
the expression of BmeIF3h at a final concentration of 0.5 mM. After shaking for an
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additional 4 h at 301C, the bacterial cells were pelleted by centrifugation (8,000 rpm,
10 min). The cells were resuspended in 15 ml of PBS (140 mM NaCl and 10 mM
sodium phosphate, pH 7.4) containing 1% Triton X-100 and were sonicated. The
soluble fractions were discarded and the inclusion bodies were denatured and
renatured according to Kuhelj et al. (1995). Briefly, the inclusion bodies were washed
twice with 20 ml of buffer A (50 mmol/L Tris–HCl, pH 8.0, 5 mmol/L EDTA), washed
twice with buffer B (950 mmol/L Tris–HCl, pH 8.0, 5 mmol/L EDTA, 2 mol/L urea),
and dissolved in 20 ml of buffer C (0.1 mol/L Tris–HCl, pH 8.0, 10 mmol/L DTT,
8 mol/L urea). Subsequently, the solution was dialyzed against 2 L of 0.1 mol/L
Tris–HCl (pH 8.0), 5 mmol/L EDTA, 5 mmol/L cysteine at 41C for 16 h. The solution
was then centrifuged to eliminate the precipitated proteins and was applied on
histidine bind resin (Amersham, Buckinghamshire, UK). rBmeIF3h was then purified
using the electroelution purification method. Purified rBmeIF3h was resolved by
12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and was
used as an antigen to immunize New Zealand white rabbits. A non-purified polyclonal
antiserum was used in all immunochemical experiments.
Immunoblotting
We isolated the epidermis, midguts, fat bodies, and silk glands from the 5th instar
larvae 0 d post-ecdysis. We also isolated ASGs, MSGs, PSGs, and fat bodies from the
feeding stage of the 3rd instar larvae (3rd-F) to the 5th instar larvae 11 d post-ecdysis
(5th–11 d), respectively. These tissues were washed, weighed, and then homogenized
in ice-cold Tris-buffered saline (TBS, 50 mM Tris-HCl pH 7.5, 150 mM NaCl)
containing protease inhibitors (1 mM phenylmethanesulfonyl fluoride). Homogenates
were centrifuged at 10,000g for 15 min, and the supernatant was collected for use in
the Western bolt analyses. Total protein quantification was performed according to
Bradford’s method (Bradford, 1976) by using bovine serum albumin (BSA) as the
standard.
Equal quantities of proteins from various tissues were separated by 12.5% SDSPAGE (Laemmli, 1970). Proteins from the same tissue were separated by 12.5% SDSPAGE and according to the protein band we adjusted the content of each protein
sample. After the proteins were electroblotted on a nitrocellulose membrane (Zhejiang
Sijia Biochemical Plastic, Zhejiang, China), they were blocked with blocking buffer (2%
non-fat milk in TBS) for 1 h. Following overnight incubation with anti-BmeIF3h serum
(1:100 dilution in blocking buffer), the membrane was washed and incubated with
peroxidase-conjugated goat-anti-rabbit IgG (Zhongshan Biotechnique, Beijing,
China). Finally, the membrane was washed thrice in TBST (0.02% Tween in TBS),
once in TBS, and incubated in a mixture of 9 ml TBS, 1 ml 4-chloro-1-naphthol in
methanol (6 mg/ml), and 6 ml H2O2 in the dark for 10 min. Subsequently, the target
band was visualized.
Hormone Treatment and Northern Blot
MSGs were dissected from the 5th instar larvae 0 h post-ecdysis and incubated with
20E (2 mM), methoprene (5 mM), and 20E (2 mM) plus methoprene (5 mM), respectively.
An equal volume of dimethyl sulfoxide (DMSO) was added to Grace’s medium as the
control. Total RNA was isolated after incubation at 3, 6, 12, 18, and 24 h and used for
Northern blot analysis.
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Eukaryotic Initiation Factor 3 Subunit H
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The ORF of the BmeIF3h cDNA was subcloned into the pGEM-T Easy vector
(Promega Biosciences, Madison, WI). The recombinant plasmid pGEM-T EasyBmeIF3h was linearized by Spe I and transcribed using T7 polymerase (Digoxigenintagged-RNA Labeling Kit; Roche, Boehringer Mannheim, Mannheim, Germany) to
synthesize the Dig-tagged antisense probe in vitro. Northern blotting was carried out
according to a procedure described previously (Zhao et al., 2002). Ten micrograms of
total RNA was briefly separated by 1% agarose-formaldehyde gel electrophoresis and
blotted onto a nylon membrane (Amersham). Subsequently, the nylon membrane was
subjected to the following treatment: UV crosslinked for 10 min, prehybridized for 2 h,
hybridized for 12 h at 681C, washed twice for 5 min each with 2 wash solution
(2 SSC, 0.1% SDS) at room temperature, washed twice for 15 min each with
0.1 wash solution (0.1 SSC, 0.1% SDS) at 681C, and blocked for 30 min at room
temperature. Finally, the signal was detected with nitroblue tetrazolium chloride/
5-bromo-4-chloro-3-indolyl phosphate.
Immunohistochemistry
MSGs and PSGs were dissected from the 4th-HCS, 5th instar larvae at 0 d (5th–0 d)
and 2 d (5th–2 d) post-ecdysis, respectively. After fixing for 16 h in 4% paraformaldehyde at 41C, the silk glands were dehydrated with a graded series of ethanol,
embedded in paraffin wax, and cut into 7-mm-thick slices. The sections were treated
according to the method described previously (Wang et al., 2007, 2009). Slides were
incubated with proteinase K (20 mg/ml) for 15 min at 371C and then blocked in 2%
BSA. Subsequently, the slides were incubated with a primary polyclonal antibody
against BmeIF3h diluted to 1:100 and then with a goat anti-rabbit-ALEXA 488
antibody (Eugene, OR) diluted to 1:1,000 in 1 PBS with 2% BSA for 2 h at room
temperature. Counterstaining was conducted with 40 , 6-diamidino-2-phenylindole
dihydrochloride (DAPI, San Jose, CA) for 10 min to show the nuclei. Negative controls
were simultaneously subjected to the same treatment, but pre-immune rabbit serum
was used instead of the antiserum against BmeIF3h. Olympus BX51 fluorescence
microscope was used to detect fluorescence.
All experiments for the studies were independently repeated three times.
RESULTS
cDNA Cloning of BmeIF3h
The complete ORF of BmeIF3h cDNA was amplified with specific primers eIF3h pET F
and eIF3h pET R based on the sequence from the National Center for Biotechnology
Information (NCBI). The BmeIF3h ORF contains 1,014 nucleotides capable of
encoding a 337–amino acid polypeptide with a predicted molecular mass of
38.6 kDa and pI of 5.68. Domain structure analysis revealed that the deduced protein
had a JAB/MPN domain, which is essential for translation initiation (Fig. 1). BLASTP
and GenBank analyses indicated that BmeIF3h was highly similar to invertebrate
eIF3h proteins and relatively less similar to vertebrate homologues. For instance,
BmeIF3h had 70% identity with the eIF3h protein of Drosophila melanogaster, and
shared 52% identities with the eIF3h proteins of Gallus.
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Figure 1. Nucleotide and deduced amino acid sequence of BmeIF3h. The JAB/MPN domain is shadowed
(aa 20–152). The translation initiation site is in bold, and an asterisk indicates the stop codon.
Recombinant Expression and Purification
Two bands, a 43-kDa band and a 30-kDa band, appeared in the purified proteins.
According to the molecular mass, the 43-kDa band, which included a 5-kDa His-tag
Archives of Insect Biochemistry and Physiology
Eukaryotic Initiation Factor 3 Subunit H
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Figure 2. Coomassie brilliant blue staining analysis of the expression and purification of rBmeIF3h fusion
protein by using 12.5% SDS-PAGE. Lane 1, crude protein extracts of bacteria of non-induction control;
lane 2, crude extracts of bacterial cells induced by IPTG for 4 h; lanes 3 and 4, soluble and insoluble protein
fractions of bacterial cells induced for 4 h, respectively; lane 5, rBmeIF3h protein purified by histidine bind
resin chromatography after denaturation and refolding; M, protein standard.
Figure 3. Expression profiles of BmeIF3h in various tissues dissected from the 5th instar larvae 0 d postecdysis (A) and each part of the silk glands (B). A, a: RT-PCR to show the expression of BmeIF3h in various
tissues at 5th–0 d; b: RT-PCR to show the actin as RNA quantity and quality control; c: Western blot to show
the expression of BmeIF3h in various tissues. Ep, epidermis; Mg, midguts; Fb, fat bodies; Sg, silk glands.
B: Western blot; ASG, anterior silk glands; MSG, middle silk glands; PSG, posterior silk glands; M, protein
standard. 5th–0 d, 5th–2 d, 5th–6 d, and 5th–11 d are the developmental stages of the silk glands.
and 38-kDa target protein, was the target protein of rBmeIF3h. The other one was a
co-purified non-specific product (Fig. 2).
Tissue Distribution of BmeIF3h
The results of the RT-PCR and Western blot demonstrated that BmeIF3h proteins
were distributed in both fat bodies and silk glands, with considerable strong signals
detected in silk glands (Fig. 3A). Furthermore, protein extracts of the different sections
of the silk glands were analyzed by Western blot. The results revealed that ASGs,
MSGs, and PSGs all expressed BmeIF3h proteins at 5th–0 d and 2 d post-ecdysis,
decreasing until 5th–11 d. Compared with the expression levels of BmeIF3h from
different parts of the silk glands dissected from the 5th instar larvae 0 d and 2 d postecdysis, respectively, we found that BmeIF3h was higher in MSGs and PSGs than in
ASGs during either period (Fig. 3B).
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Expression Profiles of BmeIF3h During Developmental Stages
Western blot analysis showed that the expression of BmeIF3h in ASGs was at a lower
level, compared to the other gland sections, although there was some increase from the
5th–0 d to the 5th–8 d. In contrast, the expression level of BmeIF3h proteins in MSG
obviously increased from the 4th–F to the 5th–4 d and then decreased until it was
hardly detected once it entered the 5th instar larvae 6 d post-ecdysis. The expression
level of BmeIF3h proteins in PSG also increased from the 4th–F to the 5th–6 d and
then decreased to lower protein levels until the 5th-11 d. However, the expression of
BmeIF3h proteins in fat bodies was different from the silk glands, which maintained a
relatively high level from the 3rd instar feeding stage to the 5th instar 2-d larvae postecdysis, dramatically decreasing when it entered the 5th instar 4-d post-ecdysis (Fig. 4).
Hormonal Regulation of BmeIF3h
To understand the hormonal regulation manner on the expression of BmeIF3h gene in
the silk gland, the variation of the expression of BmeIF3h in MSGs was examined after
incubating the silk glands in the 20E and methoprene, respectively. Northern blot
analysis revealed that the expression level of BmeIF3h was not affected by 20E except
for a little increase at 3 h after incubating with 20E. However, the expression level of
the BmeIF3h gene was obviously down-regulated after incubation with methoprene.
After incubation with 20E plus methoprene, the expression levels of BmeIF3h were
between that incubated with 20E and with methoprene alone (Fig. 5).
Figure 4. Temporal expression profiles of BmeIF3h in the ASGs, MSGs, and PSGs as well as in Fb. ASG,
anterior silk glands; MSG, middle silk glands; PSG, posterior silk glands; Fb, fat bodies. F, feeding;
M, molting. HCS, head capsule slippage.
Figure 5. Hormone regulation of the expression of BmeIF3h in MSGs. 5th–0, natural 5th instar 0 h worm;
3, 6, 12, 18, and 24 h are the durations after incubation. Ten mg of total RNA was used in each lane; 18S
ribosome RNA was used as a quantitative control.
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Eukaryotic Initiation Factor 3 Subunit H
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Cellular Localization of BmeIF3h in MSGs and PSGs
Immunohistochemistry revealed that BmeIF3h proteins were distributed in the
cytoplasm of MSG cells during the periods developed from the 4th–HCS to the 5th–2 d
(Fig. 6A). However, at the same stage, BmeIF3h was located in both the cytoplasm
Figure 6. Cellular localization of BmeIF3h in the MSGs (A) and PSGs (B) from different developmental
stages. Cross-sections of MSGs/PSGs dissected from 4th–HCS larvae (A–A’’’, D–D’’’), 5th–0 d (B–B’’’, E–E’’’),
and 5th–2 d (C–C’’’, F–F’’’), respectively. A–F: Negative controls with preserum for A’–F’, respectively. A’–F’:
BmeIF3h (green) stained by anti-BmeIF3h serum and Alexa 488. A’’–F’’: Same samples as in A’–F’ but
stained with DAPI to show the nuclei (blue). A’’’–F’’’: Merged images for Alexa 488 and DAPI. The crosssections were magnified 10 40 under the microscope. Bars 5 100 mm. Nu, nucleus; Cy, cytoplasm.
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and nuclei of PSG cells, with prominent stronger signals detected in the nuclei than in
the cytoplasm of PSG cells at the 5th–2 d (Fig. 6B).
DISCUSSION
The MSGs and PSGs function exclusively to synthesize the silk glue proteins and silk
fibre proteins, respectively, while the ASGs serve as ducts to transport the silk proteins
(Dhawan and Gopinathan, 2003). The high expression levels of BmeIF3h in MSGs
and PSGs at the 4th instar and the last instar larvae might be associated with silk
protein synthesis. Some silk protein genes like Ser2 were found to begin expression at
a high level once the larvae entered the feeding stage of the 4th instar larvae (Couble
et al., 1987). The expression levels of BmeIF3h in MSGs and PSGs were up-regulated
dramatically when the larvae entered the 4th instar feeding stage. This might be
associated with the high expression of the silk proteins.
Silk glands grow maximally during the last instar, and the synthesis of silk protein
is proportional to the gene dosage until spinning (Dhawan and Gopinathan, 2003).
However, the expression of BmeIF3h in MSGs was maintained at a high level until the
5th instar 4-d post-ecdysis and could hardly be detected after the larvae entered the
5th instar 6-d post-ecdysis when silk glue proteins were produced greatly. One possible
reason for this is that BmeIF3h did not regulate the expression of silk glue proteins
directly, but it was through the initiation of some earlier factors involved in the
translation of silk glue proteins. Kim et al. (2004) also demonstrated that Arabidopsis
eIF3h was not essential for general protein translation, but it could control the
translation of specific mRNA.
BmeIF3h was distributed in the cytoplasm of MSGs cells, but it was distributed
both in the cytoplasm and in the nuclei of PSGs cells, with a much stronger signal in
the nuclei at 2 d post-ecdysis. It was found that several other eIFs, such as eIF4E
(Dostie et al., 2000), eIF4G (McKendrick et al., 2001), eIF5A (Rosorius et al., 1999),
eIF5C (Dong et al., 2009), and eIF3f (Shi et al., 2003), expressed both in the cytoplasm
and in the nuclei. The different location of BmeIF3h in MSGs cells and PSGs cells
suggested that it had different actions related to the translation of silk glue proteins
and silk fibre proteins. Given the basal and low expression level of BmeIF3h in ASGs
during the periods detected, BmeIF3h was likely to be involved as well in the general
protein translation responsible for the growth of ASGs.
Tsuzuki et al. (2001) demonstrated that BmeIF3h/EN10 in the ASGs was
up-regulated at 8 h after stimulation by 20E, and it could be classified as a delayed
early gene of PCD. Our results indicated that the expression of BmeIF3h in
MSGs could be slightly stimulated by 20E but only within 3 h. Otherwise, juvenile
hormone analog methoprene would suppress the expression of BmeIF3h in the silk
glands.
In sum, BmeIF3h was prominently expressed in silk glands. The expression levels
of BmeIF3h in MSGs and PSGs were higher than that in ASGs. The expression levels
of BmeIF3h in MSGs and PSGs were up-regulated once the silk glands began to
synthesize silk protein. 20E slightly elicited the expression of MSGs BmeIF3h, while
methoprene suppressed the expression of the gene. BmeIF3h had different actions in
the MSGs related to the production of the silk glue proteins, and in the PSGs related to
the silk fibre proteins, respectively.
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ACKNOWLEDGMENTS
We thank Dr. Ya-Ping Zhang and Yin-Yu Gu of the Sericultural Research Institute of
Shandong Province, Yantai, China, for providing the silk worm strain.
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Archives of Insect Biochemistry and Physiology
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