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Expression of Manduca sexta V-ATPase genes mvB, mvG and mvd is regulated by ecdysteroids

Stephan Reineke, Helmut Wieczorek and Hans Merzendorfer^*

Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, 49069 Osnabrück, Germany

View larger version (27K): [in a new window]   Fig. 1. Transcript levels for V-ATPase subunits from Manduca sexta during starvation. (A) Pooled total RNA from the midgut of three fifth-instar larvae (2 µg each) was separated by gel electrophoresis, transferred onto nylon membranes and hybridized with digoxigenin-labelled ssRNA probes specific for the indicated V-ATPase subunits. Labelled transcripts from starved (left) or feeding (right) larvae were visualized by chemiluminescence and autoradiography. The far left column shows molecular mass standards (in kb). (B) Pooled total RNA was dotted onto nitrocellulose and hybridized, and chemiluminescence signals were quantified densitometrically. Values (± S.E.M., N=3) are given as a percentage of signal intensities obtained from feeding larvae and are normalized to the transcript levels of ribosomal protein S7 (Jiang et al., 1996). Representative dots have been inserted in the corresponding columns with RNA from feeding larvae in the upper row and RNA from starved larvae in the lower row. A—H, subunits of the V1 complex; a—e, subunits of the Vo complex.

View larger version (33K): [in a new window]   Fig. 2. Transcript levels of genes for V-ATPase subunits B (mvB), G (mvG) and d (mvd) from Manduca sexta during the moult. Pooled total RNA from the midgut of three larvae (2 µg each) at the stages indicated was dotted onto nylon membranes and hybridized with digoxigenin-labelled ssRNA probes. Chemiluminescence signals were quantified densitrometrically. In the upper part of the graph, values (± S.E.M., N=3) are given as a percentage of signal intensities obtained from feeding larvae and normalized to the transcript levels of ribosomal protein S7 (Jiang et al., 1996). In the lower part of the graph, published ecdysteroid and juvenile hormone titres are shown at the corresponding moulting stages (Baker et al., 1987; Bollenbacher et al., 1981; Fain and Riddiford, 1975). F4, fourth-instar day 2; MA, moulting stage A; MD, moulting stage D (approximately 20 h after MA); MF, moulting stage F (approximately 20 h after MD); F50, fifth-instar day 0; F53, fifth-instar day 3.

View larger version (20K): [in a new window]   Fig. 3. Effect of 20-hydroxyecdysone and juvenile hormone III on transcript levels of Manduca sexta V-ATPase genes mvB, mvG and mvd. 20-Hydroxyecdysone (20-HE) (200µg; approximately 0.4 mg ml-1 haemolymph) or juvenile hormone III (JHIII) (20µg; approximately 40µg ml-1 haemolymph) was injected into the dorsal vessel of fifth-instar larvae (days 2-3, 2 g body mass). After the indicated period of rearing in the presence of food, total RNA was extracted from the midgut. Pooled total RNA (2 µg) was dotted onto nylon membranes and hybridized with digoxigenin-labelled ssRNA probes. Chemiluminescence signals were quantified densitometrically. Values (± S.E.M., N=3) are given as a percentage of signal intensities obtained from untreated animals and are normalized to the transcript levels of ribosomal protein S7 (Jiang et al., 1996).

View larger version (142K): [in a new window]   Fig. 4. Immunolabelling of the V1 complex in midgut goblet cells from Manduca sexta. Cryosections from feeding (A; fifth-instar, days 2-3), starving (B; fifth-instar, day 2-3), moulting (C; fourth- to fifth-instar, moulting stage D) and 20-hydroxyecdysone-treated (D; fifth-instar, days 2-3, 24h after hormone injection) larvae were stained with monoclonal antibody 221-9 to V-ATPase subunit A (Klein et al., 1991). Scale bar, 30µm; a, apical; b, basal; g, goblet cell cavity; asterisks indicate goblet cell apical membranes; arrows point to goblet cell nuclei.

View larger version (44K): [in a new window]   Fig. 5. Overall promoter structures of the Manduca sexta V-ATPase genes mvB, mvG and mvd. Hybridization screening of a genomic -Fix II library from M. sexta led to the isolation of three genes encoding V-ATPase subunits B, G and d. The corresponding 5' upstream regions were subcloned and sequenced. Fragments of 1 kb are shown, with special emphasis on overall promoter structures in the region close to the translational start site (shaded blue). GC content was determined using a window size of 30 bp; GC contents of more than 50 % are in red, of between 20 % and 50 % are in grey and of less than 20 % are in blue. Black arrows, canonical TATA boxes; red arrows, cAMP-responsive, elements (CREs); blue arrows, ecdysterone response elements (EcREs).

View larger version (40K): [in a new window]   Fig. 6. Determination of transcriptional start sites of the Manduca sexta V-ATPase genes mvB, mvG and mvd. The start sites were determined by RNase protection assays using 973 bp each of the 5' upstream regions as a template for in vitro transcription of 32P-labelled ssRNA probes. After hybridization with poly(A) RNA, single-stranded RNA was digested with RNase A and T1. The remaining double-stranded RNA was separated on a denaturing polyacrylamide gel. Visualization of radioactively labelled bands was performed by exposing the gels to X-ray film. Nucleotide positions of identified transcriptional initiation sites that are similar to the consensus sequence KCABHYBY (Bucher, 1990) are shown on the right side of the figure.

View larger version (18K): [in a new window]   Fig. 7. Influence of 20-hydroxyecdysone on transcriptional activities of the mvB, mvG and mvd 5' upstream regions. 976 bp of the 5' upstream regions was ligated in front of the firefly luciferase gene of the pGL2-basic vector. Thus, expression of the luciferase gene was brought under the control of the V-ATPase gene promoters. Sf21 cells were co-transfected with these constructs and pR1-CMV, a control vector that constitutionally expresses Renilla luciferase and allows internal standardization. Expression of both luciferases was monitored luminometrically. The effect of the steroid hormone on reporter gene expression was determined after incubation of the transfected Sf21 cells with 2.5 µg ml-1 20-hydroxyecdysone for the times indicated. Values (± S.E.M., N=3) are given as relative units compared with untreated cells and are normalized to the luciferase activity of the control vector.

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