Expression of Manduca sexta V-ATPase genes mvB, mvG and mvd is regulated by ecdysteroids
Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, 49069 Osnabrück, Germany
* Author for correspondence (e-mail: merzendorfer{at}biologie.uni-osnabrueck.de )
Accepted 7 February 2002
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Summary |
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Key words: vacuolar H+-translocating ATPase, V-ATPase, Manduca sexta, promoter, transcription, ecdysteroid
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Introduction |
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The plasma membrane V-ATPase from the midgut of the tobacco hornworm
Manduca sexta (Lepidoptera, Sphingidae) is made up of eight different
V1 and four different Vo subunits with presumed
stoichiometries of A3B3CDEFG3H and
ac6de, respectively
(Merzendorfer et al., 2000).
In the larval midgut, the V-ATPase is present at high levels in the apical
membrane of goblet cells, where it exclusively energizes all secondary active
transport processes across the epithelium. As a result of the absence of
functional anion channels, a transmembrane voltage in excess of 250 mV is
generated, and this drives the electrogenic exchange of H+ for
K+ (Wieczorek et al.,
1991
). The combined action of the V-ATPase and the
K+/2H+ antiporter leads to net K+ secretion
and thus to a transepithelial K+-motive force that drives the
absorption of amino acids via K+-coupled amino acid
symporters (Castagna et al.,
1998
). Because of the stoichiometry of the antiporter, the gut
lumen is alkalized to a pH of more than 11, the most alkaline value produced
by any biological system (Azuma et al.,
1995
).
Regulation of V-ATPases may encompass many diverse mechanisms such as
oxidation of -SH groups or control via activator or inhibitor
proteins (Merzendorfer et al.,
1997a). During the larval/larval moult and periods of starvation,
the insect plasma membrane V-ATPase is downregulated by the reversible
dissociation of the enzyme into its V1 and Vo complexes
(Sumner et al., 1995
;
Gräf et al., 1996
). As a
result of this disassembly, cytoplasmic levels of the V1 complex
increase, a fact that incidentally allowed its efficient purification and the
first structural studies of the V1 complex
(Svergun et al., 1998
;
Grüber et al., 2000
). For
economical reasons, it appears plausible that, during periods when V-ATPase
activity is shut down, biosynthesis of V-ATPase subunits is downregulated
concomitantly. We have therefore measured the levels of transcripts and the
transcriptional activities of several V-ATPase genes. Here, we show that
transcript levels of V-ATPase subunits decrease gradually during starvation
and the larval/larval moult. Moreover, we provide evidence that the control of
transcript levels for V-ATPase genes is mediated by ecdysteroids, a class of
steroid hormone known to control larval development.
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Materials and methods |
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Cloning of the 5' regions of mvB, mvG and
mvd
To isolate the gene encoding V-ATPase subunit B (mvB), the
Manduca gDNA library was screened by a plaquehybridization procedure
(Sambrook et al., 1989). A
digoxigenin-labelled DNA hybridization probe was generated by polymerase chain
reaction (PCR) using the cDNA primers 5'-ATGGCAAAAACCCTATCCGC-3'
(positions 57-76) and 5'-CATGATCATCCAGCACAGAC-3' (positions
678-659) and, as a template, the cDNA clone encoding V-ATPase subunit B
(Novak et al., 1992
).
Hybridization and stringency wash steps were carried out at 68 °C.
Repeated isolation of positives plaques led to two independent phage clones,
-mvB1 and
-mvB2. After
Southern blotting of the phage DNA, which had been isolated from
-mvB2 according to Sambrook et al.
(1989
) and cleaved using
different restriction enzymes, a 4.1 kb NcoI fragment containing
mvB upstream sequences was identified. This DNA fragment was cloned
into pBluescript KS(), which had been modified before by blunt-end
ligation of an NcoI-linker (Stratagene) into the SmaI site
of the multiple cloning site. Cloning of the upstream regions of mvG
and mvd was performed using a similar approach.
The primer sets employed for probe synthesis were
5'-CTAAGATCGATGCTGAGACC-3'/5'-TGTCATGTGACAAAGTGGCGCT-3'
(cDNA positions 256-275/523-502 of subunit G)
(Lepier et al., 1996) and
5'-TTACTTGAACTTGGTGCAAT-3'/5'-TTCAATGTACCTACATACAG-3'
(cDNA positions 168-188/1644-1624 of subunit d)
(Merzendorfer et al., 1997b
),
respectively. Hybridization screening at 68 °C led to the isolation of
several independent phage clones for both genes
(
-mvG/dx). Following restriction pattern analysis,
Southern blotting and hybridization with appropriate probes, a
-mvG1-derived ClaI-fragment of 4 kb and a
-mvd1-derived PstI/SacI-fragment
of 1.7kb were identified as regions carrying corresponding upstream gene
sequences. Both fragments were subcloned in pBluescript KS() and
sequenced.
Reporter gene assays
The promoter activities of the mvB, mvG and mvd 5'
upstream regions were determined with the Dual Luciferase Reporter Assay
(Promega) using the pRL-CMV vector as an internal standard for transfection
efficiency. To construct the reporter gene plasmids, PCR fragments comprising
a region of 973 bp upstream of the start codons of mvB, mvG and
mvd were ligated into the SacI and HindIII cloning
sites of pGL2-basic vector. Amplification was performed in the presence of the
pBluescript KS() plasmids containing genomic fragments of mvB,
mvG and mvd. Primer pairs containing 6 bp nonsense nucleotides
at their 5' ends followed by either a SacI site in the case of
the forward primers or a HindIII site in the case of the reverse
primers were as follows:
5'-TACTCAGAGCTCAAATTTGGCATAGGCATGGC-3'/5'-TACTCAAAGCTATAGGGTTTTTGCATT-3'
for mvB;
5'-TACTCAGAGCTCTGCGAATCTTCCGTCAC-3'/5'-TACTCAAAGCTTCATGTGTCTGACTCGCCATT-3'
for mvG; and
5'-TACTCAAGCTCAGCATATCTCGTTTTTTCGA-3'/5'-TACTCAAGATCTAAATATGCAGCCCTTT-3'
for mvd. After ligation of the
SacI/HindIII-digested PCR fragments, the resulting
mvB/G/d-pGL2-basic constructs were checked by sequencing. The
corresponding reporter gene plasmid and the pRL-CMV control plasmid (2µg of
each) were co-transfected into Sf21 cells using 5 µl of Cellfectin
(Life Technologies) and an incubation period of 12 h at 27 °C. Further
steps of the assay were performed according to the manufacturer's manual.
Luminescence signals were measured in a Lumat LB 9507 luminometer (EG&G
Berthold, Germany) and specified as relative light units normalized to the
Renilla luciferase expression of pRL-CMV.
Determination of transcriptional start sites
RNA hybridization probes complementary to the 5' upstream regions of
mvB, mvG and mvd were synthesized by in vitro
transcription, as described previously
(Merzendorfer et al., 2000).
The 973 bp upstream fragments of the mvB/G/d-pGL2-basic constructs
were excised and ligated into pBluescript KS(+) using the restriction enzymes
SacI and HindIII for the mvB and the mvG
constructs, respectively, and SacI and BamHI for the
mvd construct. The resulting plasmids (1.5 µg of each) were
linearized with SacI at their 3' cloning sites, purified on an
agarose gel and used as template DNA for in vitro transcription,
which was performed with T3 polymerase at 38°C for 40 min in the presence
of 3 MBq of [
-32P]CTP (Amersham Pharmacia Biotech; 30 TBq
mmol l-1). Subsequently, the template DNA was degraded by DNase I
(Roche Diagnostics) treatment. Nucleotides that had not been incorporated were
separated from the labelled transcripts by centrifugation through Sephadex G25
spin columns (Sambrook et al.,
1989
). RNA integrity was checked by agarose gel electrophoresis
and Radient Red (BioRad) staining.
Radioactive transcripts (9x105 cts min-1) and 6 µg of the target mRNA isolated from the midgut of fifth-instar larvae were coprecipitated with sodium acetate and ethanol and resuspended in hybridization buffer consisting of 40 mmol l-1 Pipes (pH 6.4), 400 mmol l-1 NaCl, 1 mmol l-1 EDTA and 80% formamide. Hybridization was carried out overnight at 50°C. Single-stranded RNA was degraded by treatment with RNase A and T1 at 30°C for 30 min. After inactivation of the RNase by proteinase K treatment, double-stranded RNA hybrids were extracted with phenol/chloroform/isoamyl alcohol and coprecipitated with 5 µg of yeast tRNA. The denatured RNA probes were separated on a 6% polyacrylamide gel containing 7 moll-1 urea. Autoradiography was performed by exposing the gel to X-ray film (Kodak X-omat AR) for 14 days at -70°C using the BioMax TranScreen HE intensifying screen system (Kodak).
Immunohistochemistry
Larval midguts were dissected, and the gut contents were removed. After
excision of the longitudinal muscles, the tissue was stretched, cut into small
pieces of approximately 5 mm2 and fixed for 90 min at room
temperature in PLP fixative [0.1 moll-1 sodium
m-periodate, 75 mmol l-1 L-lysine, 2% (w/v)
paraformaldehyde in 0.1 moll-1 Sørensen phosphate buffer, pH
7.4]. Tissue embedding, cryosectioning and immunostaining were performed as
described previously (Klein et al.,
1991). To label the V1 complex, cryosections were
treated with the monoclonal antibody 221-9 to subunit A of Manduca
sexta V-ATPase (Klein et al.,
1991
). Visualization of the primary antibody was performed with
Cy3-conjugated anti-mouse F(ab')2 fragments (Sigma). To test
for nonspecific binding of the secondary antibody, control reactions were
carried out without primary antibodies. The sections were covered with Mowiol
(Aventis, Germany) and viewed with an Olympus IX70 fluorescence microscope. To
visualize Cy3 emission, the SWG filter set (Olympus) and monochromatic
excitation at 535 nm were used.
Other methods
Manduca sexta was reared under long-day conditions (16 h:8 h L:D
photoperiod) at 27°C using a synthetic diet for the larvae, modified
according to Bell and Joachim
(1974). Total RNA and mRNA were
prepared from the larval midgut of different developmental stages using Qiagen
RNA purification kits according to the manufacturer's protocol. Northern blots
were performed as described previously
(Merzendorfer et al., 1997b
),
except for the use of CPD-Star (Roche Diagnostics) as a chemiluminescence
substrate. RNA levels were quantified densitometrically using the Fluor-S
Multi-Imager and Quantity One software (Biorad). Chemiluminescence signals on
X-ray film (Kodak) were scanned, and the intensities were measured in units of
optical densityxmm2. Sequencing was performed on both DNA
strands using the Sequenase 2.0 Kit (Amersham Pharmacia Biotech) and following
published protocols. Several nucleotide sequences were obtained from the
custom sequencing service of MWG-Biotech. Hormone treatment of caterpillars
was carried out by injection of 20-hydroxyecdysone [20-HE; 40 µl, 5 µg
µl-1 phosphate-buffered saline (PBS) containing 10% (v/v)
2-propanol] or juvenile hormone III [JH; 40 µl, 0.5 µg
µl-1 PBS containing 10% (v/v) methanol] into the dorsal vessel.
Control animals were injected with 40 µl of the solvent.
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Results |
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To investigate whether levels of V-ATPase subunit transcripts depend on
food intake, we performed a series of northern blots. Total RNA was isolated
from 16 h starved and from feeding larvae, dotted onto nylon membranes and
hybridized with ssRNA probes for V-ATPase transcripts. Hybridization signals
were quantified densitometrically and normalized to the amounts of ribosomal
protein S7 mRNA (Jiang et al.,
1996). Starvation of fifth-instar larvae (days 2-3) resulted in
decreased transcript levels for all the V-ATPase subunits except for subunit D
(Fig. 1). Our findings suggest
that, upon starvation, both the activity of the V-ATPase and the biosynthesis
of most V-ATPase subunits are downregulated.
|
To address the question of whether levels of V-ATPase transcripts are also
downregulated during the moult, we determined the time courses for transcript
levels of subunits B, G and d, each encoding a subunit from a different part
of the holoenzyme complex: B from the V1 head, G from the
V1 stalk and d from the membrane-bound Vo portion
(Wieczorek et al., 2000).
Total RNA was isolated from the midguts of larvae at different fourth- to
fifth-instar moulting stages, classified according to Baldwin and Hakim
(1991
). Transcript levels were
detected and normalized as described above. The mRNA levels of these subunits
decreased during the moult until stage D was reached and increased to control
levels when the larvae started to feed again at the early fifth instar
(Fig. 2).
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Development and metamorphosis of insects are strictly controlled by two
major hormone classes; ecdysteroids and juvenile hormones. Increased titres of
ecdysteroids such as 20-hydroxyecdysone (20-HE) induce moulting by initiating
a regulatory cascade beginning with the activation of primary response genes.
In contrast, the function of juvenile hormones (JH) is to prevent
metamorphosis and to regulate reproductive maturation in the adult.
Interestingly, the changes in transcript levels for V-ATPase subunits B, G and
d turned out to be negatively correlated with previously published hormone
titres of 20-HE (Baker et al.,
1987; Bollenbacher et al.,
1981
) and positively correlated with those of JH
(Fain and Riddiford, 1975
;
Hiruma et al., 1999
).
Transcript levels of V-ATPase subunits reached their minima at moulting stage
D, when the haemolymph titre of 20-HE exhibits its maximum level. Conversely,
V-ATPase transcript levels started to recover in moulting stage F, when JH
titres in the haemolymph are highest. Our results suggest that either 20-HE or
JH may be responsible for the observed changes in transcript levels of
V-ATPase subunits during the moult.
Injection of 20-hydroxyecdysone leads to decreased levels of V-ATPase
transcripts and influences midgut morphology
To identify the hormone responsible for the change in RNA levels, we
injected either 20-HE or JHIII into the dorsal vessel of fifth-instar larvae
(days 2-3). Since haemolymph titres of both hormones are extremely low at this
developmental stage (Baker et al.,
1987; Bollenbacher et al.,
1981
; Fain and Riddiford,
1975
; Hiruma et al.,
1999
), injection should mimic the effects of endogenous 20-HE and
JH, as has been shown previously (Edgar et
al., 2000
; Hewes and Truman,
1994
). Caterpillars were exposed for either 6 or 24h to 200 µg
of 20-HE, 20 µg of JHIII or equal volumes of control solution containing
the corresponding solvent. They were then dissected, total RNA was isolated
from the midgut and transcript levels of subunits B, G and d were quantified
as described above. JHIII had, at the most, only a slightly negative effect on
transcript levels. In contrast, 20-HE led to a short-term increase and a
long-term decrease in transcript levels for all three subunits
(Fig. 3).
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The results of 20-HE injection are in line with the assumption that ecdysteroids are involved in the regulation of levels of V-ATPase transcripts during the moult. To evaluate the effects of 20-HE injection on midgut morphology and the intracellular localization of V1 complexes, we performed an immunohistochemical study comparing cryosections of posterior midguts from larvae treated with 20-HE for 24h with those from moulting (fourth larval moult, stage D), starving (16h, fifth-instar, days 2-3) and control (feeding, fifth-instar, days 2-3) larvae (Fig. 4). In comparison with midguts from feeding larvae, the midgut cells from 20-HE-treated, moulting and starving larvae appeared to be elongated and the goblet cavities appeared to be reduced in diameter.
|
To visualize the V1 complex, we used the monoclonal antibody
221-9 to subunit A of the V-ATPase (Klein
et al., 1991). In midguts of feeding animals, the signal for
V-ATPase subunit A was almost exclusively located in the region of the goblet
cell apical membrane. In contrast, 20-HE-treated, moulting and starved larvae
exhibited significantly different staining patterns. In all three cases, the
antibody to subunit A labelled the cytoplasm of the goblet cells intensively,
indicating detached V1 complexes, as has been demonstrated
previously for moulting and starved larvae using biochemical approaches
(Sumner et al., 1995
;
Gräf et al., 1996
). These
experiments clearly demonstrated that injection of 20-HE leads to changes in
general midgut morphology and in the subcellular distribution of V1
complexes as they are also observed during moulting and starvation.
Upstream regions of V-ATPase genes mvB, mvG and mvd differ in general
structure but all contain ecdysone response elements
The injection experiments suggested that the control of transcript levels
encoding V-ATPase subunits may be mediated by ecdysteroids. Since steroid
hormones are known to be potent regulators of transcriptional activity, we
cloned and sequenced the 5' upstream regions of mvB, mvG and
mvd, the Manduca sexta genes encoding V-ATPase subunits B, G
and d, respectively. Our aim was to compare the promoter structures and to
investigate the influence of ecdysteroids on the promoter activities. The
nucleotide sequences were compared over a region of approximately 1 kb
upstream of the translational start codon
(Fig. 5).
|
Although dissimiliar in sequence, the promoters of mvB and
mvG shared features common with inducible or tissue-specific
promoters of vertebrates. They showed canonical TATA boxes and a low GC
content of approximately 30 %, with a similiar distribution pattern in the
proximal region. In addition, both promoters contained a motif similar to the
consensus sequence of the cAMP-responsive element (CRE)
(Roesler et al., 1988). In
contrast, the promoter of mvd lacked apparent TATA boxes and CREs and
exhibited a different GC distribution pattern, although the averaged GC
content of approximately 35 % was only negligibly higher. Overall,
mvd appeared to exhibit several characteristics common to
housekeeping genes described in other organisms. However, all three promoters
contained an ecdysone-responsive element (EcRE) corresponding to the consensus
sequence KNTCANTNNMM (Luo et al.,
1991
).
Thus, the EcREs found in the 5' regions of mvB, mvG and
mvd may act as common transcriptional regulator elements that, upon
the release of ecdysteroids, simultaneously control the promoter activities of
different V-ATPase genes in a concerted fashion. This interpretation is in
line with the observed decline in levels of V-ATPase transcripts during the
moult and upon 20-HE injection. To characterize the promoters further, we
mapped the transcriptional start sites of the mvB, mvG and
mvd 5' regions by RNase protection assays using
32P-labelled RNA probes covering the corresponding upstream
sequences between nucleotide positions -973 and +1. As shown in
Fig. 6, analysis of the
autoradiograms revealed four transcriptional start sites for mvB, one
for mvG and six for mvd, all of which were similar to the
CAP consensus sequence KCABHYBY (Bucher,
1990). Thus, the upstream regions of mvB and mvd
exhibit multiple transcriptional start sites, whereas the upstream region of
mvG contains only a single start site, although there was a second
protected fragment at a very distal position but no corresponding CAP site in
close proximity.
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Transcriptional activities of mvB, mvG and mvd promoters are
inhibited by ecdysterone
To test the effect of 20-HE on the transcriptional activities of V-ATPase
genes, we performed reporter gene assays in Sf21 insect cells. We
ligated 976 bp (positions -973 to +3) of the mvB, mvG and
mvd upstream sequences into pGL2-basic, a vector using firefly
luciferase as the genetic reporter. Normalization of transfection efficiency
was achieved by co-transfection with pRL-CMV, which contains a CMV promoter to
provide constitutive expression of Renilla luciferase. After
co-transfection of Sf21 cells with the corresponding reporter gene
plasmid, we added 20-HE to the culture medium and incubated the cells for
different times. After cell harvest and lysis, we successively measured the
luminescence signals derived from firefly and Renilla luciferase. All
upstream sequences cloned in front of the luciferase coding sequence led to
significant transcriptional activities, suggesting that all these regions
actually contain promoter sequences that allow binding of basic transcription
factors and of RNA polymerase II.
After addition of 2.5µg ml-1 20-HE to the culture medium, a decrease in promoter activities for mvB, mvG and mvd was observed after 3, 5 and 48h, suggesting that ecdysteroids inhibit the transcription of V-ATPase genes (Fig. 7). In contrast, short-term treatment for only 1.5 h led to significant activation of the mvB and slight activation of the mvG promoter (Fig. 7). Except that short-term activation was not detected for the mvd promoter, these findings were similar to the results obtained for the 20-HE injection experiments, in which a shortterm increase and a long-term decrease in transcript levels were observed.
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Discussion |
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Northern blots showed decreased transcript levels for almost all
investigated V-ATPase subunits upon starvation and during the moult. These
findings suggest that the larvae are very economical in dealing with their
anabolic resources since they downregulate V-ATPase subunit synthesis during
periods when no enzyme activity is needed because of the cessation of food
intake. Since transcript levels correlated with the haemolyph titres of 20-HE
and JHIII, we injected both hormones into feeding fifth-instar larvae at times
when haemolymph titres of both hormones are known to be very low. In contrast
to JHIII, which had no effect, injection of the moulting hormone 20-HE
resulted in a short-term increase and a long-term decrease in levels of
V-ATPase transcripts, implying that either transcription rates or transcript
stabilities are regulated by the steroid hormone. Indeed, all 5'
upstream regions of the genes investigated, mvB, mvG and
mvd, contained putative ecdysone response elements
(Luo et al., 1991), and
reporter gene assays also demonstrated the influence of 20-HE.
Ecdysteroids are known to be key regulatory factors for gene transcription,
activating a nuclear receptor heterodimer consisting of the ecdysone receptor
EcR and the Drosophila retinoid X receptor homologue USP, the
ultraspiracle protein (Yao et al.,
1993). Ecdysteroid-mediated control of transcriptional activities
may be positive or negative depending on the hormone concentration (for a
review, see Spindler et al.,
2001
). Upregulation of transcriptional activities during insect
development by ecdysteroids is well documented in the literature. For
instance, Eips 28/29 are Drosophila genes that are
controlled tissue- and stage-specifically by ecdysone-responsive elements
present in the upstream and downstream flanking regions
(Andres and Cherbas, 1994
).
Other genes that are likely to be regulated by the activated ecdysterone
receptor are the Drosophila genes encoding the yolk protein
(Bownes et al., 1996
), the
heat-shock proteins hsp23 and hsp27 (Luo
et al., 1991
) and the caspase DRONC
(Dorstyn et al., 1999
;
Hawkins et al., 2000
) and the
M. sexta genes EcR-A and EcR-B1, which encode two
ecdysone receptor isoforms (Jindra et al.,
1996
).
Downregulation of transcriptional activities by ecdysteroids was observed
too, but there was no evidence for direct transcriptional repression. For
instance, it has been suggested that the ecdysteroid-regulated gene
esr20, which is expressed in the trachea of M. sexta, is
downregulated at ecdysis, probably because of a decline in transcript
stability that might be triggered indirectly by 20-HE
(Meszaros and Morton, 1997).
Characterization of the dopamine decarboxylase gene (DDC) of M.
sexta revealed that it may be indirectly suppressed by 20-HE via
an ecdysteroid-induced transcription factor that itself suppresses DDC
transcription (Hiruma et al.,
1995
).
From our experiments, we conclude that 20-HE influences V-ATPase gene
expression in more than one way. Injection of 20-HE into fifth-instar larvae
(days 2-3) resulted in both a short-term increase and a long-term decrease in
V-ATPase transcript levels. At first sight, this seems to be contradictory
because V-ATPase activity is not initially upregulated upon moulting or
starvation. However, pump deactivation appears not to be directly related to
transcriptional control of subunit synthesis because reassembly of the
V1 and V0 complex has been shown previously to be
independent of biosynthesis in yeast and in M. sexta
(Kane, 1995;
Merzendorfer et al., 1997a
).
Thus, short-term upregulation of transcription does not necessarily lead to
upregulation of V-ATPase activity, especially since translation rates do not
have to follow transcription rates strictly.
In contrast to 20-HE, injection of JHIII had only negligible effects on
transcript levels. However, we cannot exclude the possibility that treatment
with other JH isoforms could influence mRNA levels of V-ATPase subunits
because, in lepidopterans, the haemolymph titres of JHI and JHII are
significantly higher than that of JHIII
(Baker et al., 1987). Results
similar to those of the injection experiments were obtained in reporter gene
assays with upstream regions of the V-ATPase genes mvB, mvG and
mvD, indicating that transcription rates are influenced by 20-HE in
both directions, depending on the duration of 20-HE treatment. In principle,
the decrease in V-ATPase gene expression could be due to a general repression
of transcript levels, as has been observed in M. sexta during the
fifth instar between days 2 and 3 and upon ecdysteroid treatment of day 1
epidermis by RNA labelling experiments
(Shaaya and Riddiford, 1988
).
However, we believe the decrease to be specific since we normalized all our
assays either to ribosomal S7 mRNA or to the expression of a constitutive
promoter.
The observation that levels of V-ATPase transcripts also decline during
starvation independent of moulting processes suggests that, during these
periods, ecdysteroids may be involved in the control of transcript levels.
Unfortunately, to our knowledge, ecdysteroid titres have not been measured in
the haemolymph of starving larvae. However, there might be a further level of
control that is dependent upon feeding and independent of edysteroids, as was
suggested for the Drosophila melanogaster yolk protein gene
transcription (Bownes et al.,
1988). Thus, both the lack of nutrients and the rising titre of
ecdysteroids may contribute to the decrease in V-ATPase mRNA levels upon
starvation and during the moult. This resembles the expression pattern of
arylphorin in M. sexta, where both the lack of nutrients and the
rising ecdysteroid titre contribute to the decrease in arylphorin mRNA levels
during moults and during the wandering stage
(Webb and Riddiford,
1988
).
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Acknowledgments |
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References |
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