Cloning and characterization of CcEcR. An ecdysone receptor homolog from the Mediterranean fruit fly Ceratitis capitata

Cloning and characterization of CcEcRAn ecdysone receptor homolog from the Mediterranean fruit fly Ceratitis capitata

Meletis Verras, Manolis Mavroidis, Giorgos Kokolakis, Polyxeni Gourzi, Antigone Zacharopoulou andAnastassios C. Mintzas

Division of Genetics, Cell and Developmental Biology, Department of Biology, University of Patras, Greece

In order to understand the role that 20-hydroxyecdysone plays during development of the Mediterranean fruit fly

Ceratitis capitata (medfly), a major agricultural pest, we have cloned a Ceratitis ecdysone receptor (CcEcR) and

studied its expression and its binding properties to an ecdysone response element. Using the conserved DNA

binding region of the Drosophila melanogaster ecdysone receptor (DmEcR) B1 cDNA as a probe, we

isolated a medfly cDNA clone containing the coding region, a part of the 5 0-untranslated region and the

complete 3 0-untranslated region of a CcEcR. The deduced CcEcR polypeptide contained all five domains typical

of a nuclear receptor. Alignment comparisons and phylogenetic analyses indicated that CcEcR most closely

resembled the B1 isoform of DmEcR and Lucilia cuprina EcR homolog (LcEcR) relative to all other known

ecdysone receptors. In situ hybridization analysis showed that the CcEcR gene is mapped in the region 53B of the

4R chromosome arm, while Northern hybridization analysis showed that CcEcR transcripts have a size of

approximately 8 kb. Significant levels of CcEcR transcripts were detected in eggs, middle and late embryos, late

third instar larvae and middle prepupae. The levels of the CcEcR transcripts during the other larval stages as well

as during pupal and adult stages were much lower, while during the early stages of embryogenesis were very low.

Electrophoretic mobility shift assays indicated that CcEcR binds specifically to the Drosophila hsp27 ecdysone

response element as a heterodimer with Drosophila USP, the product of the ultraspiracle gene. Our structural and

biochemical data suggest that CcEcR is the functional homolog of the B1 isoform of DmEcR.

Keywords: ecdysone; ecdysone receptor; nuclear receptor; medfly; Ceratitis capitata.

The steroid hormone 20-hydroxyecdysone functions as atemporal signal to coordinate multiple developmental eventsduring the life cycle of the insects by altering the expression ofa large number of genes. Like other regulators of development,including steroids, thyroid hormones, retinoids, and vitamin D,ecdysone controls gene transcription by binding to a nuclearreceptor which acts as a ligand-dependent transcription factor[1±5]. The ecdysone receptor (EcR) is homologous to thethyroid hormone, vitamin D and retinoic acid receptors andbinds to target DNA response elements (EcREs) as aheterodimer with the product of the ultraspiracle gene (USP),the insect homolog of the mammalian retinoid X receptor [5±10]. The ecdysone receptor of the Drosophila melanogaster(DmEcR), was the first ecdysone receptor that was cloned andcharacterized [11]. Following that, DmEcR homologs havebeen cloned and sequenced for a number of insect species aswell as for an acarine and a crustacean species ([3] for a review,and [12±16]). The last decade, a large number of ecdysone-regulated genes have also been cloned and characterized inD. melanogaster [4,17,18]. Genetic, molecular and bio-chemical studies on these genes and their products have

confirmed the ecdysone-regulated chromosome puffing modelof Ashburner [19], and have extended it to all cells and tissuesundergoing either histolysis or differentiation and morpho-genesis during metamorphosis. Parallel studies in other species,are of interest in order to verify the basic mechanisms ofecdysone regulation and reveal potential important differencesamong species. Such studies are particularly important ininsects of economic importance as they could lead to thedevelopment of novel species-specific control methods [20].

We are interested in studying ecdysone regulation in theMediterranean fruit fly Ceratitis capitata (medfly), a wide-spread and destructive pest of the soft fruit [21]. This higherdipteran species has a similar life cycle to D. melanogaster andpresents several important advantages for studying ecdysoneregulation. It has been adapted easily to laboratory culture, haswell characterized salivary gland polytene chromosomes [22]and can be staged precisely during the last hours of larvaldevelopment, because of the jumping event that takes placeabout 6 h before puparium formation [23]. C. capitata is thebest studied fruit fly species of economic importance at thegenetic and molecular level. During the last years, significantgenetic information has been accumulated for this species[24±27] and several genes have been cloned and mappedon the polytene chromosomes [28,29]. Furthermore, twogermline transformation systems have been recently developedin the medfly [30,31] thus facilitating basic molecular geneticstudies and opening up the possibility for genetic control ofnatural medfly populations.

In a previous report, we presented a detailed analysis of thesalivary gland polytene chromosome puffing patterns found on

Eur. J. Biochem. 265, 798±808 (1999) q FEBS 1999

Correspondence to A. C. Mintzas, Department of Biology, University of

Patras, Patras 26500, Greece. Fax: + 30 61 997881/994797,

Tel.: + 30 61 997368, E-mail: [email protected]

Abbreviations: medfly, Mediterranean fruit fly; CcEcR, Ceratitis capitata

ecdysone receptor; DmEcR, Drosophila melanogaster ecdysone receptor;

BmEcR, Bombyx mori ecdysone receptor; J, jamping; Pf, puparium

formation; USP, ultraspiracle gene;

Note: EMBL sequence data bank under the accession number Y08954.

(Received 16 April 1999, revised 4 August 1999, accepted 11 August 1999)

q FEBS 1999 Medfly ecdysone receptor (Eur. J. Biochem. 265) 799

the five autosomes of the medfly during larval and prepupaldevelopment [23]. Most of these puffs have been characterizedin vitro by tissue culture experiments in the presence ofecdysone and protein synthesis inhibitors (Gariou-PapalexiouA., Mintzas A. & Zacharopoulou A., unpublished results). Inthis study, we have cloned and analyzed a cDNA from Ceratitiscapitata encoding a homolog of the B1 isoform of DmEcR aswell as characterized its expression throughout medfly develop-ment and its binding properties to an ecdysone responseelement.

M A T E R I A L S A N D M E T H O D S

Animals

The Benakeion mass rearing strain of C. capitata, having thestandard gene arrangement [22], was used in this study. Thisstrain has been maintained in our laboratory for more than20 years under standard conditions (25 ^ 1 8C, 40±50%relative humidity and 12 h light: 12 h dark photoperiod) aspreviously described [32].

General methods

Genomic DNA was prepared from 24-h-old embryos accordingto the protocol described by Ashburner [33]. Preparation ofphage DNA, agarose gel electrophoresis and blotting toNytran-plus membranes (Schleicher & Schuell) were carriedout using standard procedures [34]. DNA probes wereprepared by random hexanucleotide priming. Hybridizationsof [32P]-labeled probes to blotted DNA fragments wereperformed as described by Sambrook et al. [34], at 658(D. melanogaster probe) or at 68 8C (C. capitata probes) in6 � NaCl/Cit, 0.5% SDS, 200 mg´mL21 sonicated, heat-denatured salmon sperm DNA and 5 � Denhardt's solution.Filters were washed twice at room temperature in 2 � NaCl/Cit, 0.1% SDS for 15 min each, and twice at 55 8C in1 � NaCl/Cit, 0.1% SDS for 15 min each. Filters withhomologous probes, were further washed at 65 8C in1 � NaCl/Cit, 0.1% SDS for 30 min.

cDNA Library screening

A medfly cDNA library, kindly provided by C. Savakis (IMBB;constructed by S. Brogna), was used for screening. The librarywas constructed in Lamda ZapII (Stratagene) using poly(A)+

RNA from whole larvae at the late third larval instar.Approximately 4 � 105 plaque forming units were screenedby hybridization at 65 8C, as described by Benton & Davis[35], using as a probe the DNA binding region of theD. melanogaster EcR cDNA [11]. Hybridization and washeswere performed under the same conditions used for Southernblot analysis with the Drosophila probe. Positive clones werepurified to homogeneity and converted into pBluescriptSKplasmids by the in vivo excision method [36] using the R408helper phage according to the manufacture's instructions(Stratagene). A PCR-amplified fragment from one of theseclones, containing the putative A/B and C regions of a medflyEcR, was then used to screen the same cDNA library underhigh stringency conditions. Hybridization and washes wereperformed under the same conditions used for Southern blotanalysis with homologous probes. The positive clones from thisscreen were isolated, converted into pBluescriptSK plasmids asabove and characterized by restriction mapping, Southern blotanalysis and sequencing.

DNA sequencing and computational analysis

Overlapping fragments of the medfly cDNAs in pBluescript(Stratagene) were sequenced from both strands by thedideoxynucleotide chain termination method [37] using[a-35S] dATP and the USB Sequenase version 2.0 kit(Amersham). Computer-assisted sequence analysis was per-formed using the pc/gene (Inteligenetics) software package.Database searches were performed at NCBI using the blastnetwork service [38]. Multiple sequence alignments wereperformed using the clustal x program with defaultparameters [39]. The CcEcR cDNA sequence has beensubmitted to the EMBL data bank with accession numberY08954.

In situ hybridization

Squash preparations of salivary gland polytene chromosomeswere made from 5-day-old larvae. Hybridization was per-formed by using biotin-16UTP (Boehringer Mannheim) forDNA labeling as previously described [28] with a minormodification: the Avidin Elite kit (Vector Laboratories)was used for signal detection instead of HorseradishPeroxidase.

Northern blot and RT-PCR analysis

Total RNA was isolated from whole animals using the urea/phenol/chloroform method of Holmes & Bonner [40]. ForNorthern analysis, 20 mg of total RNA samples were separatedby electrophoresis on 1.3% agarose gels containing formalde-hyde and then transferred to Nytran-plus membranes [34].Hybridization and washes were performed under the sameconditions used for Southern blot analysis with homologousprobes.

To evaluate the CcEcR mRNA levels throughout medflydevelopment, we performed RT-PCR analyses using total RNAfrom synchronized animals of different developmental stages.Maximum synchronization of the medfly cultures was obtainedaccording to the procedure described by Gariou et al. [23].Following this procedure, the embryonic, larval and prepupalstages last 49 ^ 1, 151 ^ 2 and 40 h, respectively, while thepupal stage lasts about 8 days. Adults were synchronized bycollecting newly hatched flies within a period of 10 min. Theamount and purity of each RNA sample was estimated usingspectrophotometric measurements at 260 and 280 nm. Theconcentration of the RNA in all samples was verified bycomparing the intensity of the ribosomal RNA bands inethidium bromide stained gels and was readjusted propor-tionally when necessary. Reverse transcription and polymerasechain reaction were carried out in a single tube using the AccessRT-PCR system of Promega, according to the manufacturer'sinstructions and the products were analyzed on 1.5% agarosegels and stained with ethidium bromide. The two specificprimers used (P1 and P2, Fig. 2), were designed to anneal tosequences in exons on opposite sides of an intron in order todifferentiate between amplification of cDNA and amplificationof contaminating genomic DNA. In preliminary experiments, aseries of dilutions for each RNA sample were used in order todetermine subsaturation conditions for the PCR products. In thefinal experiments, 200 ng of RNA from each stage were used in100 mL reactions which were processed simultaneously for 40amplification cycles.

800 M. Verras et al. (Eur. J. Biochem. 265) q FEBS 1999

In vitro transcription, translation and electrophoreticmobility shift assays

pBluescript plasmids containing the coding regions forC. capitata EcR and D. melanogaster EcR and USP wereused for in vitro transcription. The two Drosophila plasmidswere kindly provided by F. Kafatos (EMBL): The EcR plasmidcontained the full length cDNA of the B1 isoform ofDrosophila EcR [11] excluding the first 621 residues of the5 0-untranslated region and the USP plasmid was the pXR2C8cDNA clone [41]. Coupled transcription and translation werecarried out using the Promega TNT T7/T3 coupled reticulocytelysate system according to the manufacture's instructions.Circular plasmid DNA (1 mg) was used as a template in 50 mLreactions with T7 or T3 polymerase at 30 8C for 2 h. Thesamples were then placed on ice or were stored at 280 8C untilused. Initial reactions were performed in the presence of1 mCi [14C]leucine and the products were analyzed bySDS/PAGE and fluorography. The DNA binding reactionswere conducted as described bellow. Unlabeled TNT extracts(3±5 ml) were incubated at room temperature for 15 min in15 ml total volume of a buffer containing 50 mm KCl, 30 mmHepes (pH 7.9), 1 mm dithiothreitol, 1 mm MgCl2, 0.3 mmEDTA, 4% glycerol and 100 ng´mL21 poly(dI-dC). Eachreaction was incubated for another 15 min following additionof 0.02 pmol of labeled hsp27-EcRE [42]. The probe wasprepared by annealing the two oligonucleotides, 5 0-AGAGA-CAAGGGTTCAATGCACTTGTCC-3 0 and 5 0-ATTGGACAA-GTGCATTGAACCCTTGTCTCT-3 0, according to Kadonaga& Tjian [43] and was labeled with [a-32P] dATP to specificactivity about 8 � 107 cpm´mg21 by fill-in reaction withKlenow enzyme. The reactions were then loaded into 4%nondenaturing polyacrylamide gel in 0.25 � Tris/borate/EDTArunning buffer. After electrophoresis, the gel was dried andexposed to X-ray film at 280 8C with an intensifying screen.For the competition experiments, 1 pmol of unlabeled hsp27-EcRE was added at the same time with the labeled probe. Forthe supershift experiments, 2 mL of the anti-USP monoclonalantibody AB11 [44], provided by F. Kafatos, were added at theend of the binding reactions and the reactions were incubatedfor 40 more min at 4 8C.

R E S U L T S

Isolation and characterization of a medfly cDNA cloneencoding the homolog of the B1 isoform of the DrosophilaEcR

In order to clone the ecdysone receptor (EcR) of C. capitata,we first screened a cDNA library from late third instar medflylarvae, under stringent conditions, using as a probe a 421-bpEcoRI/SacI fragment isolated from a cDNA clone of D.melanogaster EcR (see Materials and methods). This probe,containing the entire DNA binding domain of the Drosophila

EcR [11], gives single hybridizing bands in Southern blotanalysis of restricted medfly genomic DNA under the samehybridization conditions (data not shown). Five stronglyhybridizing clones, containing inserts of 1±1.3 kb, wereisolated from a screen of 4 � 105 plaque forming units.Restriction mapping and partial sequencing of the inserts,indicated that the clones were overlapping in a 1-kb region.Complete sequencing of one of the largest inserts (C1), shownin Fig. 1, revealed that it had an open reading frame of 390amino acids encoding a polypeptide with significant homologyto the B1 isoform of D. melanogaster EcR and other insecthomologs. Amino acid sequence comparisons suggested thatthis polypeptide included the entire A/B (activation) and C(DNA binding) domains as well as part of the D (hinge) domainof a putative medfly EcR. A PCR-amplified fragment from theC1 clone, containing the putative A/B and C regions, was thenused to screen the same cDNA library under highly stringentconditions. Several strongly hybridizing clones were isolatedfrom a screen of 4 � 105 plaque forming units and werepartially characterized by restriction mapping, Southern blotanalysis and sequencing. One of these clones, named CM6, hadan insert of approximately 3.2 kb which completely overlappedwith the C1 cDNA and contained a short poly(A) tail at the 3 0end. The restriction mapping of this clone is shown in Fig. 1.Complete sequencing of the CM6 cDNA on both strands,indicated that this cDNA includes the complete coding regionof a C. capitata EcR (CcEcR).

Figure 2 shows the nucleotide sequence and the deducedamino acid sequence of CcEcR cDNA. The nucleotidesequence includes a part of the 5 0-untranslated leader sequenceand a long 3 0-untranslated sequence, containing a putativepolyadenylation signal centered at position 3103. The deduced

Fig. 1. Schematic representation of C. capitata ecdysone receptor

(CcEcR) (top) and restriction maps and arrangement of C1 and CM6

EcR cDNAs (bottom). Letters above EcR denote the receptor domains and

numerals below indicate the number of amino acids in each domain

determined by multiple amino acid sequence alignments among all known

insect EcRs (top). C1 is a cDNA obtained by screening a medfly cDNA

library, under stringent conditions, with a probe containing the entire

DNA binding region of the Drosophila ecdysone receptor [11]. CM6 is

a cDNA obtained by screening the same library, under highly stringent

conditions, with a fragment from the C1 cDNA clone containing the A/B

and C regions of CcEcR (bottom).

Fig. 2. Nucleotide sequence and the deduced amino acid sequence of a CcEcR as determined from the sequences of the C1 and CM6 EcR cDNAs

shown in Fig. 1. The DNA sequences of the C (DNA binding) and E (hormone binding) domains are underlined. The cysteine residues of the zinc finger

motifs of the C domain are underlined. The P-box residues (E266 to G270) and the D-box residues (K285 to S289) which are important for the specific

recognition of the hormone response element of a particular gene [52] are in bold. Two heptapeptide sequences which are potential nuclear localization

signals [53] are underlined in the D (hinge) domain. A putative helix-turn-zipper motif in the E (ligand binding) domain is underlined. Putative amino acids

contributing to the heterodimerization [54] are indicated by asterisks. A putative polyadenylation signal (AATAAA) is in bold letters and underlined.

Nucleotides corresponding to the sense (P1) and antisense (P2) primers used in RT-PCR analysis are shaded. The EMBL data bank accession number for this

sequence is Y08954.



Fig. 2. continued.


amino acid sequence starts with a double methionine atnucleotide 265 and ends with a stop codon at nucleotide2286. It must be noted that neither of the initial methionineswas found in a context with a strong match to the Drosophilaconsensus sequence for translation initiation [45]. The openreading frame contains 673 codons that potentially encode apolypeptide with a predicted molecular weight of 73.74 kDa.This polypeptide exhibits the five domain structure (A/B, C, Dand F) that is characteristic of all members of the nuclearreceptor family [1,2]. The P-box (E266 to G270) of CcEcR is100% identical to the P-boxes of all other EcRs and members ofthe thyroid hormone/retinoic acid receptor subfamily while theD-box (K285 to S289) shows 80% identity to Drosophila EcRD-box and 60% to the D-boxes of all other dipteran andlepidopteran EcRs.

We have compared the deduced amino acid sequence ofCcEcR with the respective amino acid sequences of the B1isoforms of D. melanogaster, Manduca sexta, Bombyx mori andTenebrio molitor EcRs as well as with the sequences of eightother EcR homologs. Multiple alignment of these sequences,revealed that all show high degree of similarity with very highamino acid identity in the C and E domains (data not shown).The level of identity for each domain (A/B to F) of each of the12 EcRs, with respect to the corresponding domain for CcEcR,is shown in Table 1 along with the peptide length for eachdomain. In these calculations, a small number of nonaligningN-terminal residues of B. mori and Heliothis virescens EcRswere not included in the alignments. Figure 3A shows anindicative dendrogram generated by the alignment of the Edomains, the sequences of which have been determined in allEcRs cloned today. Overall these data indicate that CcEcR ismost similar to the other dipteran EcRs and less similar to theEcRs from lepidopterans, Locusta migratoria, T. molitor andother noninsect arthropod species. Among the dipteran EcRs,LcEcR and DmEcR appear the most strongly related to CcEcR

thus forming one group, while AaEcR and CtEcR are moredivergent. The dipteran EcRs also show significant similarity intwo segments of the A/B domain (Fig. 3B). The N-terminal andthe C-terminal sequences in this domain (aa 1±53 and aa199±247 in CcEcR) have been well conserved among the fourdipteran EcRs. The amino acid identity in this domain betweenCcEcR and the B1 isoform of DmEcR is 52% while thesimilarity reaches 72%. The respective similarity to the otherDrosophila EcR isoforms was negligible (results not shown).These data suggest that CcEcR can be classified as ahomologue of the B1 isoform of Drosophila EcR. The Fdomain is very variable in length and amino acid sequenceamong all insect EcRs. This variability is more pronounced inDiptera than in Lepidoptera.

Chromosomal localization of CcEcR gene

When a PCR-generated 120 bp portion of the DNA bindingdomain of CcEcR was used as a probe in Southern blot analysisof restricted medfly genomic DNA (data not shown), singlehybridization bands were observed for all digests suggestingthat CcEcR is encoded by a single gene. The cytologicalposition of this gene was determined by in situ hybridization tosalivary gland polytene chromosomes from 120-h-old larvae,using the full length CM6 EcR clone as a probe (Fig. 4). Asingle hybridization signal was observed in the region 53B onthe 4R chromosome arm [22]. This region corresponds to anecdysone regulated puff which is active in 120-hr-old larvaeand forms a Balbiani-ring-like structure [23].

Northern hybridization analysis and developmentalexpression of CcEcR transcripts

A 750-bp PCR-amplified fragment encoding the E region ofCcEcR (thus expected to detect all CcEcR isoforms) was used

Table 1. Comparison of the predicted amino acid sequences among ecdysone receptor homologs.

Length (amino acids) % Identitya

Region A/B C D E F Total A/B C D E F Total

Ccb 247 66 92 221 47 673

Dmc 263 66 101 221 227 878 52 95 67 94 11 57

Lcd 300 66 87 221 83 757 49 95 78 95 23 67

Aae 189 66 92 222 106 675 41 94 58 85 12 57

Ctf 114 66 109 222 25 536 14 95 52 75 15 48

Bmg 146 66 91 223 20 546 31 94 30 69 11 48

Msh 146 66 92 224 28 556 29 94 37 71 15 50

Cf i 140 66 91 223 19 539 30 94 46 72 , 10 50

Hvj 146 66 97 220 31 560 29 92 42 71 21 51

Tmk 127 66 74 219 4 491 29 88 38 68 , 10 47

Lml 179 66 74 219 3 541 27 95 40 69 , 10 47

Upm ± o 66 74 218 4 ± ± 89 35 59 , 10 ±

Aamn 178 66 94 219 ± 560 25 86 32 62 ± 43

a Identity versus C. capitata ecdysone receptor homolog (CcEcR), expressed as percentage of the consensus length. b Ceratitis capitata ecdysone receptor

(Diptera). c Drosophila melanogaster ecdysone receptor isoform B1 [11] (Diptera). d Lucilia cuprina ecdysone receptor homolog [13] (Diptera). e Aedes

aegypti ecdysone receptor homolog [47] (Diptera). f Chironomus tetans ecdysone receptor homolog [48] (Diptera). g Bombyx mori ecdysone receptor isoform

B1 [49] (Lepidoptera). A small number of nonaligning N-terminal residues extended beyond the initiation methionine of the other EcRs, were not included.h Manduca sexta ecdysone receptor isoform B1 [50] (Lepidoptera). i Choristoneura fumiferana ecdysone receptor homolog [12] (Lepidoptera). j Heliothis

virescens ecdysone receptor homolog [GeneBank accession No. Y09009] (Lepidoptera). A small number of nonaligning N-terminal residues extended

beyond the initiation methionine of the other EcRs, were not included. k Tenebrio molitor ecdysone receptor isoform B1 [14] (Coleoptera). l Locusta

migratoria ecdysone receptor homolog [51] (Orthoptera). m Uca pugilator ecdysone receptor homolog [16] (Decapoda). n Amblyomma americanum

ecdysone receptor homolog (EcRA1) [15] (Ixodida). o The sequences of these regions have not yet been defined.


as a probe in Northern hybridization analysis. Figure 5A showsthat total RNA from late third instar larvae gives a majorhybridizing band of approximately 8 kb. A major band ofsimilar size was also detected in total RNA from embryos andadults (results not shown), suggesting that CcEcR transcriptshave very long 5 0 untranslated leader sequences. The abun-dance of EcR transcripts in all developmental stages of themedfly was estimated by RT-PCR, using two specific primers(P1 and P2) from the D and E regions of the CcEcR cDNAshown in Fig. 2. These primers were expected to amplify acommon region of all receptor isoforms. When these primerswere used to amplify medfly genomic DNA and CcEcR cDNA,

two different bands of approximately 600 and 320 bp,respectively, were obtained (results not shown), indicatingthat the CcEcR gene contains approximately 280 bp intronsequence(s) between these two primers. Two hundred nano-grams of total RNA from precisely staged embryos, larvae,prepupae, pupae and adults were analyzed simultaneously asdescribed in Materials and methods. The results from arepresentative RT-PCR experiment are shown in Fig. 5B.CcEcR transcripts are abundant in freshly laid eggs anddecrease to minimal levels in the first 6 h of embryogenesis.During the following 6 h, transcript levels increase reaching amaximum in 9- to 12-h-old embryos. Thereafter they show a

Fig. 3. An indicative dendrogram of the

aligned amino acids sequences of the hormone

binding (E) domains from 11 insect and two

other arthropod ecdysone receptors, and

comparison of the amino acid sequences of the

N-terminal transactivation (A/B) domain

among dipteran ecdysone receptor homologs.

(A) Conceptually translated sequences were

aligned using the clustal x program with

default parameters [39]. The sequence of the E

domain of D. melanogaster USP [41] was used

as an outgroup. Bootstrap values (1000

replicates) are indicated on the nodes of the

Bootstrap N-J Tree. The sequences were obtained

from the Genbank data base as indicated in

Table 1. (B) Asterisks, double dots and single

dots denote fully conserved, strongly conserved

and weakly conserved residues, respectively.


moderate decrease and remain relatively constant till the endof embryogenesis. In the first five days of larval development,the EcR transcript levels are relatively low and constant. Duringthe last 10 h of larval development, they show a gradualincrease reaching maximum levels around jamping (J), acharacteristic event that occurs about 6 h before pupariumformation (Pf) [23]. At puparium formation, a stage that marksthe onset of pupariation [46], the EcR transcript levels decreasesignificantly and remain low for the following 18 h while they

increase again during the second half of the prepupal stage.During the pupal and adult stages, the EcR transcripts don'tshow significant changes. The developmental profile of the EcRtranscripts during late larval and prepupal stages is in goodagreement with the activity pattern of the 53B puff, where theEcR gene is mapped, and is related with the overall puffingactivity in the salivary gland polytene chromosomes [23] (seeDiscussion).

CcEcR binds specifically to Drosophila hsp27-EcRE as aheterodimer with Drosophila USP

To test whether CcEcR binds to ecdysone response elements(EcREs), we carried out gel mobility shift assays using in vitrosynthesized Ceratitis EcR and D. melanogaster EcR andultraspiracle (DmUSP) proteins as described in Materials andMethods. The in vitro synthesized proteins were tested byelectrophoresis on SDS-polyacrylamide gels. As shown inFig. 6(A), in vitro transcription/translation of the CcEcR cDNAyielded a major product with a molecular mass of approxi-mately 82 kDa. The D. melanogaster EcR and USP cDNAsalso yielded single protein products of the expected size.Figure 6B, shows the results from a gel mobility shift assay inwhich the Drosophila hsp27-EcRE was used as a probe.DmEcR was used as positive control. As was expected, neitherof the three proteins by itself bound to the hsp27-EcRE. WhenDmUSP was added, the CcEcR bound to the EcRE and formeda major complex of similar intensity to the respective DmEcRcomplex. The CcEcR complex runs faster than the DmEcRcomplex and this is in agreement with the smaller size ofCcEcR comparatively to DmEcR. The CcEcR complex appearsto be specific as its formation was completely prevented byadding 50-fold excess of unlabeled EcRE. Furthermore, thiscomplex was supershifted when a monoclonal anti-USPantibody was added to the reaction mixture. These data indicatethat CcEcR binds specifically to the Drosophila hsp27-EcRE as

Fig. 4. Chromosomal localization of the CcEcR gene. Salivary gland

polytene chromosomes from 5-day-old larvae were hybridized with the full

length CM6 EcR clone as described in Materials and methods. A single

hybridization signal was observed in the region 53B on the right arm of

chromosome 4. The centromere region of this chromosome is indicated

by C.

Fig. 5. Northern hybridization analysis of

CcEcR mRNA and developmental expression

of CcEcR transcripts. (A) Twenty micrograms

of total RNA from late third instar larvae were

fractionated by formaldehyde agarose gel

electrophoresis, transferred to nylon membranes

and hybridized with a [32P]-labeled probe

containing the E region of CcEcR. The arrow

indicates the position of the CcEcR mRNA.

(B) Two hundred nanograms of total RNA from

precisely staged embryos (E), larvae (L),

prepupae (PP) were analyzed simultaneously by

RT-PCR using two specific primers from the D

and E regions of the CcEcR as described in

Materials and methods. The numbers indicate the

age of embryos, larvae and prepupae in hours.

J and Pf indicate jumping and puparium

formation, respectively. P and A indicate

4-day-old pupae and adults, respectively.


a heterodimer with DmUSP and this does not require binding ofedysone.

D I S C U S S I O N

CcEcR sequence analysis

Using the DNA binding region of the Drosophila ecdysonereceptor (B1 isoform) cDNA as a probe, we have isolated a3217-bp cDNA that includes the coding region, a part of the5 0-untranslated region and the complete 3 0-untranslated regionof a transcript encoding a C. capitata ecdysone receptor(CcEcR). Sequence analysis showed that the cloned cDNAcontains a long open reading frame of 673 codons encoding apolypeptide with a predicted molecular mass of 73.74 kDa.Like Aedes aegypti and Lucilia cuprina EcRs [13,47], theCcEcR protein sequence appears to start with a doublemethionine. As neither of these potential initiation codonsconfirms to the Drosophila consensus sequence for translationinitiation [45], it cannot be postulated which of these initiationcodons is used in vivo. Amino acid sequence comparisons ofCcEcR with respective sequences of all known EcRs and othernuclear receptors, showed that CcEcR exhibits the charac-teristic domain structure of all members of the nuclear receptorfamily and belongs to the thyroid hormone/retinoic acidreceptor subfamily.

EcR isoforms that have common C and E domains butdifferent N-terminal regions, have been identified inD. melanogaster [55] as well as in several lepidopterans[12,56,57], in the mealworm T. molitor [14] and in the acarineAmblyomma americanum [15]. Comparison of the CcEcR A/Bdomain to the respective domains of the B1 isoform ofDrosophila EcR and other dipteran EcR homologs, revealedsignificant similarity in the N-terminal and C-terminalsegments of this domain indicating that CcEcR is theC. capitata homolog of the Drosophila EcR B1 isoform.Furthermore, these data suggest that sequences in the two endsof this domain may be important for the function of the B1receptor isoform. We have carried out amino acid sequencecomparisons of CcEcR with 10 insect EcRs and two otherarthropod EcRs. Our data indicate that CcEcR is most similar to

the other higher dipteran EcRs, which form one group, and lesssimilar to the EcRs from lepidopterans which form a separategroup. T. molitor, L. migratoria, A. americanum and Ucapugilator EcRs are more divergent forming two separategroups. The C, D and E domains have approximately thesame number of amino acids in all EcRs, but the A/B and Fdomains are highly variable both in length and amino acidsequence. However, as has been reported for lepidopteran EcRs[12], there is significant conservation in the A/B domain ofdipteran EcRs.

Size and expression of CcEcR mRNA

Northern hybridization analysis showed that the CcEcRtranscripts have a size of approximately 8 kb. This suggeststhat the full length CcEcR mRNA should contain a long(approximately 5 kb) leader sequence as has been reported forseveral other EcR transcripts [12,14,15,47,50,51,57].

In Drosophila, high levels of EcR transcripts were observedduring the embryonic, late larval and prepupal stages while theEcR transcript levels were much lower during the first andsecond larval instars and in adults [11,55,58]. A similar patternof expression has been reported for L. cuprina EcR transcripts[13]. We used RT-PCR assays to evaluate the EcR transcriptlevels throughout medfly development. Our results indicate thatthe developmental changes that occur in the levels of theCcEcR transcripts are very similar to those reported forD. melanogaster, especially during the late larval and prepupalstages. In freshly laid eggs, the CcEcR transcript levels are veryhigh and decrease dramatically during the first few hours ofembryogenesis. This is most likely due to the accumulation ofmaternal EcR mRNAs during oogenesis. In a previous study wehave shown that the major changes in the number and theactivity of the polytene chromosome puffs of the medfly, takeplace around jumping, at puparium formation and at the middleof the prepupal stage [23]. Furthermore, like in Drosophila, theoverall puffing activity in the medfly shows a positive cor-relation to the ecdysone titer in the haemolymph, suggestingthat most of the puff changes are regulated by ecdysone [23].As is shown in Fig. 5B, the major changes in the accumulationof the CcEcR mRNAs occur at the same developmental stages

Fig. 6. Electrophoretic analysis of Ceratitis

EcR (CcEcR) and Drosophila USP (DmUSP)

and EcR (DmEcR) on 8% SDS-polyacrylamide

gel, and electrophoretic mobility shift assays.

(A) All proteins were in vitro translated from

cDNA clones, using a coupled transcription and

translation reticulosyte lysate system as described

in Materials and methods, in the presence of

[14C]leucine. (B) In vitro translated proteins were

incubated with [32P]-labeled hsp27-EcRE of

D. melanogaster, either alone or mixed together,

and then analyzed on 4% nondenaturing

polyacrylamide gel as described in Materials and

methods. In the competition experiments, a

50-fold excess of unlabeled hsp27-EcRE was

added at the same time with the labeled probe. In

the suprshift experiment, anti-USP monoclonal

antibody AB11 [44] was added at the end of the

binding reaction.


where the major changes in puffing activity take place, that is atjumping, puparium formation and midprepupal stage. Further-more, the EcR gene maps in the region 53B which correspondsto an ecdysone regulated puff ([23] and unpublished results).These data allow us to postulate that ecdysone titer regulatesEcR levels in the medfly as has been demonstrated inD. melanogaster, M. sexta and Choristoneura fumiferana[12,56,59] and that EcR levels may play important role in theregulation of the medfly ecdysone-regulated genes.

CcEcR binds to an ecdysone response element as aheterodimer with Drosophila USP

In D. melanogaster, the functional ecdysone receptor is aheterodimer consisting of an EcR polypeptide and the productof ultraspiracle gene (USP), the insect homolog of themammalian retinoid X receptor [6±8]. This has also beendemonstrated for other species. The EcR homolog from B.mori (BmEcR) [49], has been shown to dimerize withBmUSP homolog in vitro to form an ecdysone-dependentcomplex with the hsp27-EcRE of D. melanogaster [10].Similar results have been reported for A. aegypti EcR andUSP homologs [9]. In C. fumiferana, it has been shownthat C. fumiferana EcR (CfEcR) binds to Drosophilahsp27-EcRE as a heterodimer with BmUSP, but this does notrequire binding of ecdysone [12]. We have demonstrated bymobility shift assays, that CcEcR forms a specific complex withDrosophila hsp27-EcRE only in the presence of DrosophilaUSP and that the formation of this complex does not requirehormone binding (Fig. 6). The strength of this complex wassimilar to that of the homologous DmEcR/DmUSP/hsp27-EcRE complex, suggesting that the differences in the aminoacid sequences between CcEcR and DmEcR in the C and Edomains, are not important for binding to EcRE and fordimerization to USP.

In conclusion, our data demonstrate that we have cloned afunctional ecdysone receptor (B1 isoform) from the medfly. Inother studies, we have characterized a large number of loci,including EcR, that are regulated either directly or indirectly by20-OH ecdysone ([23] and unpublished results). Furthermolecular, genetic and functional studies on the basic com-ponents involved in ecdysone regulation are necessary forunderstanding the mode of action of the hormone in thisspecies. Such studies, which are currently in progress in our lab,could reveal particular features of the mechanisms of theecdysone action in the medfly and could lead to thedevelopment of species-specific control methods for thiseconomically important pest.

A C K N O W L E D G E M E N T S

This work was supported by grants from the Hellenic General Secretariat

for Research and Technology and from the International Atomic Energy

Agency. We are grateful to Dr C. Savakis for providing the medfly cDNA

library and Dr F. Kafatos for providing the cDNA plasmids for Drosophila

EcR and USP and the monoclonal antibodies against Drosophila USP. We

also wish to thank Dr K. Mathiopoulos for reading the manuscript and

making valuable suggestions and I. Zacharopoulou for technical assistance.

Mr M. Verras would like to express his gratitude to the Greek Scholarship

Foundation for a postgraduate Scholarship.

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Documents

Cloning and characterization of CcEcR. An ecdysone receptor homolog from the Mediterranean fruit fly Ceratitis capitata