The ultraspiracle Gene of the Spruce Budworm,Choristoneura fumiferana: Cloning of cDNAand Developmental Expression of mRNASRINI C. PERERA,1,2 SUBBA R. PALLI,1* TIM R. LADD,1 PETER J. KRELL,2AND ARTHUR RETNAKARAN1

1Natural Resources Canada, Canadian Forest Service, Sault Ste. Marie, Ontario P6A 5M7, Canada2Department of Microbiology, University of Guelph, Guelph, Ontario, NIG 2W1, Canada

ABSTRACT Cloning and characterization of aChoristoneura fumiferana ultraspiracle (Cfusp) cDNAare described. First, a PCR fragment and then a cDNAclone (4.4 kb) were isolated from spruce budwormcDNA libraries. Comparison of the deduced amino acidsequence of this cDNA with the sequences in Genbankshowed that this sequence had high homology with theultraspiracle cDNAs cloned from Drosophila melanogas-ter (Dmusp), Bombyx mori (Bmusp), Manduca sexta(Msusp), and Aedes aegypti (Aausp). The Cfusp cDNAcontained all the regions that are typical for a steroid/thyroid hormone receptor superfamily member. The DNAbinding domain or C region was the most conservedsequence among all the usps. The A/B, D, and E regionsalso showed high amino acid identity with the aminoacid sequences of Dmusp, Msusp, Bmusp, and Aausp.The Cfusp 4.5-kb mRNA was present in the embryos, inall larval stages, and in the pupae. The Cfusp mRNAlevels in the midgut increased during the sixth-instar larvaldevelopment and reached peak levels during the ecdys-teroid raises for the pupal molt. However, Cfusp mRNAlevels remained unchanged in the midgut of fifth-instarlarvae, and in the epidermis and fat body of sixth-instarlarvae indicating both a tissue- and stage-specific regula-tion of Cfusp mRNA expression. Dev. Genet. 22:169–179, 1998. r 1998 Wiley-Liss, Inc.

Key words: 20-hydroxyecdysone; receptor; DNA-binding; RXR

INTRODUCTIONThe steroid hormone, 20-hydroxyecdysone (20E), is

one of the major hormones that regulate molting andmetamorphosis in insects. Each molt is preceded by apulse of ecdysteroids in the hemolymph which triggersthe expression of a cascade of genes involved in themolting process [reviewed by Riddiford, 1993]. Ecdyster-oids regulate gene expression by forming a complexwith intracellular receptors, which then binds to DNAregulatory elements near the target genes. The ecdy-sone receptor complex is a heterodimer consisting of

two proteins, Ecdysone receptor (EcR) and Ultraspi-racle (USP). Both EcR and USP proteins are membersof the steroid/thyroid hormone receptor superfamily.Members of this superfamily are characterized by thepresence of four regions, A/B (transactivation region), C(DNA binding region), D (hinge region), and E (ligandbinding region) and, in some cases, region F [reviewedby Henrich and Brown, 1995; Thummel, 1995].

A cDNA that encodes EcR, a member of the steroid/thyroid hormone receptor superfamily, was first de-scribed from Drosophila melanogaster [Koelle et al.,1991]. Subsequently, EcR cDNAs from other insects,such as Chironomus tentans [Imhof et al., 1993], Man-duca sexta [Fujiwara et al., 1995; Jindra et al., 1996],Aedes aegypti [Cho et al., 1995], Bombyx mori [Sweverset al., 1995], and Choristoneura fumiferana [Kothapalliet al., 1995], were cloned and characterized. High-affinity binding of EcR to hsp27, an ecdysone-responseelement (EcRE) [Riddihough and Pelham, 1987], de-pends on heterodimerization of EcR with anothernuclear receptor, the ultraspiracle protein (USP) [Yao etal., 1992, 1993; Thomas et al., 1993]. The usp (ultraspi-racle) gene was first identified in Drosophila melanogas-ter [Henrich et al., 1990; Oro et al., 1990; Shea et al.,1990] and was shown to be homologous to the mamma-lian retinoid X receptor (RXR) [Oro et al., 1990]. Sincethen, usp cDNAs from Bombyx mori [Tzertzinis et al.,1994], Manduca sexta [Jindra et al., 1997], and Aedesaegypti [Kapitskaya et al., 1996] have been cloned. Two

Contract grant sponsor: Canadian Forest Service/Natural Sciencesand Engineering Research Council Partnership Program; Contractgrant number: CRD 0192158.

The gene bank accession number for the sequence presented isAF016368.

*Correspondence to: Subba R. Palli, Natural Resources Canada,Canadian Forest Service, 1219 Queen Street East, P.O. Box 490, SteMarie, Ontario P6A 5M7, Canada; E-mail: rpalli@nrcan.gc.ca

Received 20 October 1997; accepted 26 January 1998

DEVELOPMENTAL GENETICS 22:169–179 (1998)

r 1998 WILEY-LISS, INC.

different usp isoforms with variable A/B regions anddifferent expression profiles were identified in Man-duca sexta [Jindra et al., 1997] and in Aedes aegypti[Kapitskaya et al., 1996]. Apart from being the partnerof EcR in mediating the ecdysone response, USP in D.melanogaster is also required for normal eye morphogen-esis, fertilization, egg-shell morphogenesis [Oro et al.,1992], normal embryo development [Oro et al., 1992;Perrimon et al., 1985], and adult thoracic development[Henrich et al., 1994].

The spruce budworm, C. fumiferana, is a severedefoliator of conifers in Canada and northern parts ofthe United States. We have initiated a program todevelop a target-specific pest management strategybased on interfering with ecdysone action. We havecloned and characterized two isoforms of C. fumiferanaecdysone receptor, EcR-A (S.C. Perera, T.R. Ladd, andS.R. Palli, unpublished observations) and EcR-B [Kotha-palli et al., 1995], two isoforms of Choristoneura hor-mone receptor 3 [Palli et al., 1996, 1997a], and twoisoforms of Choristoneura hormone receptor 75 [Palli etal., 1997c; S.R. Palli, M.S. Sundaram, and T.R. Ladd,unpublished observations). This paper reports the cDNAcloning and developmental expression of C. fumiferanausp (Cfusp). In the sixth instar, Cfusp mRNA levelsincreased in the midgut during the ecdysteroid in-creases for the pupal molt. However, Cfusp mRNAlevels remained unchanged in the epidermis and fatbody of sixth-instar larvae, as well as in the midgut offifth-instar larvae, indicating both a tissue- and stage-specific regulation of Cfusp mRNA expression. The datapresented in this paper also show that the cloned CfuspcDNA can be translated into a functional usp protein,which can bind to hsp27 ecdysone response elements inthe presence of CfEcR.

MATERIALS AND METHODS

Animals

C. fumiferana eggs were collected within 1 hr afteroviposition and were kept at 22°C and 70% relativehumidity. Under these conditions the eggs hatched in 8days. After five days, the first-instar larvae molted intosecond instar and entered diapause. The diapausinglarvae were kept on cheesecloth sheets and maintainedat 2°C for 20–25 weeks to satisfy the diapause require-ment. At the end of diapause, the larvae were reared onan artificial diet at 22°C and 70% relative humiditywith a 12:12-hr light/dark photoperiod as described byGrisdale [1970]. Fifth- and sixth-instar larvae wereselected within 30 min after molting, when the headcapsules were still white, and their developmentalstages were recorded from that time onwards. Morpho-logical characteristics, such as head capsule slippage(HCS), were used as selection criteria for staging thelarvae.

RNA Extractions

Total RNA was isolated from the staged insects ortissues dissected from these insects using the guani-dine isothiocyanate-phenol-chloroform extractionmethod [Chomczyski and Sacchi, 1987]. The RNA con-centrations were determined spectrophotometrically.Poly (A)1 RNA was selected using biotin-labeled oligo(dT) and streptavidin MagneSphere TM particles (Pro-mega, Madison, WI).

Polymerase Chain Reaction

Phage DNA was isolated from a cDNA library madefrom first- and second-instar C. fumiferana larvae indiapause [Palli et al., 1997b] and used in a polymerasechain reaction (PCR) reaction with the degenerateprimers, USP3 (forward primer, 58 TGT/C GAA/G GGA/C/G/T TGT/C AAA/G GGA/C/G/T TTC/T TTC/T AA 3)8and USP4 (reverse primer, 58 TCT/C TCT/C TGA/C/G/TACA/C/G/T GCT/C TCA/C/G/T CG/TT/C TTC ATA/C/G/T CC 38) that were designed based on the conservedamino acids in the DNA binding region of Bmuspsequence [Tzertzinis et al., 1994]. The PCR cycles usedwere as follows: 94°C, 30 sec; 55°C, 30 sec; 72°C, 2 minfor 35 cycles; and 94°C, 30 sec, 55°C, 30 sec, and 72°C,10 min for the final cycle. The amplified product (170base pairs [bp]) was used to screen a cDNA library.

If Cfusp isoforms are produced similar to those in M.sexta and A. aegypti, the 58 untranslated region shouldbe unique to each isoform. An isoform-specific fragmentwas amplified from the USP2413 clone, by using thepBluescript vector primer T3 (58 ATT AAC CCT CACTAAAGG G 38) and a sequence-specific primer U268 (58CAC ACT TGA CAT GAC CAC GAA C 38). The PCRconditions used were the same as those describedabove.

cDNA Library Construction and Screening

The cDNA library was constructed in the Uni-Zap XRvector using the Zap cDNA Gigapack 11 Gold CloningKit (Stratagene Cloning Systems, LaJolla, CA) andRNA isolated from CF-203 cells (a continuous cell linedeveloped from C. fumiferana midgut). The library wasscreened with the 170-bp PCR product (amplified withUSP3 and USP4 primers), labeled with [32P] using therandom prime labeling method [Feinberg and Vogel-

=Fig. 1. Nucleotide and deduced amino acid sequence of Cfusp. Num-bers on right refer to the amino acids and those on the left to thenucleotide sequence. The A/B, C, D, and E domains (indicated on theright margin) of Cfusp protein are shown in differently shaded boxes.The different regions are identified on the basis of their similarity tothe amino acid sequence of Msusp [Jindra et al., 1997], Bmusp[Tzertzinis et al., 1994], Aausp [Kapitskaya et al., 1996], and Dmusp[Oro et al., 1990], based on Krust et al. [1986]. Arrows with a solid line,region used as the total Cfusp probe; arrows with a dotted line, regionused as the isoform-specific probe.

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Fig. 1.

stein, 1984]. The hybridization and washing of filterswere carried out as described by Palli et al. [1997b].

The usp cDNA clone was completely sequenced inboth directions by using the ALFexpress automatic

sequencer (Pharmacia Biotech, Baie d’Urfe, Quebec,Canada). Sequence analysis was performed with theMac Vector DNA analysis program (International Bio-technologies, New Haven, CT). The sequence was com-

Fig. 2. Alignment of the deduced amino acid sequence of Cfusp withother insect usps (Msusp-1 [Jindra et al., 1997], Bmusp [Tzertzinis etal., 1994], Aauspa [Kapitskaya et al., 1996], and Dmusp [Oro et al.,1990]). Consensus sequence: asterisks, conserved substitutions. Gaps

were introduced to improve the alignment. Multiple sequence align-ment was performed using the CLUSTAL program [Higgins and Sharp,1988].

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pared with other sequences in GenBank using theBLAST network service from the National Center forBiotechnology Information [Altschul et al., 1990].

Northern Blot Hybridizations

Ten micrograms of total RNA were denatured andseparated on a 1.3% formaldehyde-agarose gel andtransferred to hybond N membrane (Amersham LifeScience, Oakville, ONT, Canada). The RNA was stainedby adding 1 µg of ethidium bromide to the samplebefore denaturation. The RNA was cross-linked to themembrane using a UV cross-linker (Stratagene CloningSystems). The entire 3.7-kb insert of the shorterUSP2313 clone (total usp probe) or the PCR fragmentamplified with T3 and U268 primers (isoform-specificprobe) were random prime labeled with [32P] and usedto probe the RNA blots. Prehybridization, hybridiza-

tion, and washes were performed as described by Palliet al. [1997b].

Gel Retardation Assays

In vitro translated CfEcR and Cfusp were used for gelretardation assays. In vitro translation was carried outusing the Promega TNT-coupled reticulocyte lysatesystem, according to the manufacturer’s instructions.Gel retardation assays were carried out as described byKothapalli et al. [1995]. EcRE [ecdysone response ele-ment-58 AGA GAC AAG GGT TCA ATG CAC TTG TCCAAT 38; Riddihough and Pelham, 1987] was endlabeledusing T4 polynucleotide kinase and [g-32P] ATP (6,000Ci/mmol), and purified by passing through a SephadexG25 column. A total of 5 µl each of in vitro-translatedproteins (CfEcR and Cfusp) and 2 µl of 103 assay buffer(13 assay buffer is 10 mM Tris-HCl, pH 7.5, 50 mM

Fig. 3. Comparison of deduced amino acidsequence of Cfusp with insect usps and theirhuman homologues (Msusp-1 [Jindra et al., 1997],Bmusp [Tzertzinis et al., 1994], Aauspa[Kapitskaya et al., 1996], Dmusp [Oro et al.,1990], human RXRa [Mangelsdorf et al., 1990],and human RXRb [Chambon et al., 1992]. Thedistribution of amino acids among steroid/thy-roid hormone receptor domains (A/B, C, D, and E)according to Krust et al. [1986] are shown. Theletters in Cfusp indicate the domains of a mem-ber of the steroid hormone receptor superfamily.The numbers in Msusp-1, Bmusp, Dmusp,Aauspa, human RXRa, and human RXRb denotethe amino acid identity (top) and similarity (bot-tom) between Cfusp and Msusp-1, or Bmusp, orDmusp, or Aauspa, or human RXRa, or HumanRXRb. The percent amino acid identity and simi-larity were calculated based on the identical andsimilar amino acids between Cfusp and each ofthe other receptors using the CLUSTAL program[Higgins and Sharp, 1988].

Fig. 4. Cfusp expression during embryogenesis, and developmentof first- and second-instar larvae. A total of 10 µg of total RNA isolatedfrom embryos or larvae collected on each day of development wasseparated on a formaldehyde-agarose (1.3%) gel. The RNA was

transferred to Hybond N membrane and hybridized with the totalCfusp probe (top). Bottom, ribosomal RNA stained with ethidiumbromide. RNA sizes (in kb) from an RNA ladder (GIBCO-BRL) aregiven on the left.

Choristoneura fumiferana usp 173

NaCl, 1 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, 4%glycerol, 0.05 µg/µl poly[dI-dC] and 20 µM single-stranded, nonspecific DNA) and 7 µl of DD H2O weremixed in a 0.5-ml tube and incubated at room tempera-ture for 20 min; 1 µl (0.05 pmol) of labeled EcRE wasadded to the reaction mixture and incubated for afurther 20 min at room temperature. The componentsof the reactions were then resolved on a 6% nondenatur-ing polyacrylamide gel. The gel was fixed in 7% aceticacid, dried onto a 3M Whatman paper, and exposed tox-ray film (Hyperfilm-MP, Amersham Life Science). Incompetition experiments, 1.25 pmol of unlabeled EcREor a retinoid-related orphan receptor response element,RORE [Giguere et al., 1994] was included in the initialreaction mixture prior to adding the labeled probe.

RESULTS

Isolation and Sequencing of Cfusp cDNA

A 170-bp cDNA fragment was amplified by PCR usingtwo degenerate primers, (USP3 and USP4) designed onthe basis of amino acid sequence in the DNA bindingregion of B. mori usp (BmCF1) [Tzertzinis et al., 1994].The deduced amino acid sequence of this PCR fragmentshowed 95% amino acid identity with the correspondingamino acid sequence of Bmusp DNA binding region.This PCR fragment was then used as a probe to screen acDNA library made with the RNA isolated from CF-203cells [Palli et al., 1997b]. Two positive clones, USP2413(4.4 kb) and USP2313 (3.7 kb) were isolated. Restric-tion enzyme analysis and sequencing showed that the

shorter clone was truncated at the 58 end. Conse-quently, we used the longer clone for further analysis.

The longer cDNA clone (hereafter designated Cfusp)was completely sequenced on both strands. Figure 1shows the nucleotide sequence of the cDNA and thepredicted amino acid sequence of the longest openreading frame (ORF) of Cfusp. The longest ORF con-tained 472 codons, which translates into a 52-kDaprotein. When the Cfusp cDNA clone was translatedusing the TNT rabbit reticulocyte system in the pres-ence of 35S-labeled methionine, a 52-kDa protein wasdetected by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) (data not shown) indicat-ing that this is the ORF used. Following this ORF thereis a long (2,969-bp) 38 untranslated region, whichcontained a poly (A)1 sequence.

Figure 2 shows the alignment of the Cfusp-deducedamino acid sequence with the deduced amino acidsequences of two lepidopteran (Manduca sexta, Msuspand Bombyx mori, Bmusp) and two dipteran (Dro-sophila melanogaster, Dmusp and Aedes aegypti, Aausp)insects. The deduced amino acid sequence of Cfusp wasmore similar to the Msusp-1 isoform than to the Msusp-2isoform (data not shown). Domain comparisons be-tween Cfusp sequence and other insect usp sequences,and two mammalian usp homologues (human RXR aand b) are summarized in Figure 3. The DNA bindingregions (C domain) of all insect usps were highlyconserved, with Cfusp showing 97% identity withMsusp, and 95% identity with Bmusp, Aausp, andDmusp. The DNA binding region of Cfusp also shared

Fig. 5. Cfusp expression in second-, third-, and fourth-instar larvae. A total of 10 µg of total RNAisolated at different time intervals was analyzed by Northern Blots as described in Fig. 4. RNA sizes (inkb) from an RNA ladder (GIBCO-BRL) are given on the left.

Fig. 6. Developmental expression of CfuspmRNA in the midgut of fifth instar. Ten µg of totalRNA isolated from fifth-instar larval midgut at12-hr time intervals were analyzed by Northernblots as described in Fig. 4. RNA sizes (in kb)from an RNA ladder (GIBCO-BRL) are given onthe left.

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significant amino acid identity with the two mamma-lian RXR, human RXRa (85%) and b (82%). A highdegree of identity was also seen in the hinge (D) and

ligand binding (E) regions of Cfusp, Msusp, and Bmusp.The D region of Cfusp was 91% and 87% identical withthat of Msusp and Bmusp, respectively. The E region

Fig. 7. A: Concentration of 20E in the hemolymph during the sixthinstar (based on the data published in Palli et al. [1995]) and therelative expression of Cfusp in the midgut throughout sixth instar(relative to the expression at 144 hr after ecdysis as 100) as deter-mined by densitometric scanning of three Northern blots. The autora-diographs were converted into computer photographic files using theIS-1000 Digital Imaging System (Alpha Innotech, San Leandro, CA).The relative intensity of each band was determined using the QGelElectrophoresis Gel Imaging Analysis System (QuantiGel, Madison,WI). The mean and standard error at each time interval are shown.*Significantly different from the value at 108 hr after ecdysis to the

sixth instar (P 5 0.1, Student’s t-test). B: Tissue-specific expression ofCfusp in the midgut, epidermis, and fat body of sixth instar. Tenmicrograms of total RNA isolated at 12-hr intervals beginning atecdysis were separated on a formaldehyde-agarose (1.3%) gel, trans-ferred to Hybond N membrane and hybridized with the Cfusp probe.In the midgut: (a) hybridization with the total Cfusp probe; (b)hybridization with the isoform-specific probe. Ribosomal RNA stainedwith ethidium bromide is shown at the bottom of each panel. RNAsizes (in kb) from an RNA ladder (GIBCO-BRL) are given on the left.HCS, head capsule slippage. PP, pre-pupa.

Choristoneura fumiferana usp 175

showed 83% and 82% identities with Msusp and Bmusp,respectively. The A/B domain was the least conservedregion of all the usps. Cfusp A/B domain showed 72%and 66% identity with that of Msusp and Bmusprespectively, but was less than 33% identical with theother usps and RXRs. Interestingly, in the A/B region,there was a stretch of 13 amino acids adjacent to the Cregion, which was 100% identical in all five insect usps,but not in their human RXR homologues. Overall, theusp sequences of the two lepidopteran species, M. sextaand B. mori, showed the highest degree of conservation

with Cfusp, with 82% and 80% overall identities respec-tively, as compared to 49% for D. melanogaster and 51%for A. aegypti.

Developmental Expression of Cfusp mRNA

The developmental expression profile of Cfusp mRNAwas studied by Northern blot hybridization. Northernblots containing 10 µg of total RNA were hybridizedwith the total Cfusp cDNA probe. The Cfusp probedetected a 4.5-kb transcript in most of the tissues anddevelopmental stages tested. The Cfusp mRNA ap-peared immediately after oviposition but decreased inabundance during embryonic development (Fig. 4). TheCfusp mRNA was detected in first-instar larvae, and insecond-instar larvae that are preparing to enter dia-pause (Fig. 4), those that are in prediapause, diapause,and postdiapause (Fig. 5). Cfusp mRNA was presentand showed little variation in second-instar larvae thatcame out of diapause and resumed feeding, as well as inthird- and fourth-instar larvae (Fig. 5). Similarly, CfuspmRNA was present and showed little or no variation inits levels in the midgut tissue samples collected fromfifth-instar larvae (Fig. 6). However, in the sixth-instarlarval midgut, the Cfusp mRNA levels started to in-crease at 120 hr after ecdysis and remained high untilecdysis to the pupal stage (Fig. 7A,B). To confirm thatCfusp mRNA levels increased in the sixth-instar larvalmidgut, Northern blot analysis and Cfusp mRNA quan-titation were performed in three replications. Figure 7Ashows the mean and standard error of Cfusp mRNAlevels in the midgut of sixth-instar larvae in compari-son with ecdysteroid titre in the hemolymph. Thisincrease in the Cfusp mRNA levels coincided with theprepupal ecdysteroid peak in the hemolymph. Hybrid-ization of the same Northern blot with an isoform-specific probe (see under Materials and Methods)showed that the expression pattern of this Cfusp iso-form is similar to the total Cfusp mRNA expressionpattern, indicating that most of the increase in CfuspmRNA levels before larval–pupal metamorphosis is dueto an increase in expression of this usp isoform (Fig.7B). On the other hand, no changes in the Cfusp mRNA

Fig. 8. Expression of Cfusp during pupal development. A total of 10 µg of total RNA isolated at 12-hrintervals beginning at ecdysis were analysed by Northern blots as described in Fig. 4. RNA sizes (in kb)from an RNA ladder (GIBCO-BRL) are given on the left.

Fig. 9. Cfusp protein binds to hsp27 EcRE as a heterodimer withCfEcR. Proteins were synthesized using Cfusp or CfEcR cDNAs in theTNT system. Either CfUSP or CfEcR, or both together were incubatedwith 32P-labeled hsp27 EcRE. In competition studies 25-fold or 200-fold excess of unlabelled EcRE or unlabelled RORE (retinoid relatedorphan receptor response element) were added to the components ofthe reaction. After incubation at room temperature for 20 min, theproducts were separated on a 6% nondenaturing gel. The gel was driedand exposed to x-ray film for autoradiography.

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levels were detected in the sixth-instar larval fat body,epidermis (Fig. 7B) or the pupae (Fig. 8).

CfUSP Binds to hsp27 EcRE

USP has been shown to be a part of the functionalecdysone receptor complex [reviewed by Henrich andBrown, 1995; Thummel, 1995]. In vitro-translatedCfEcR and Cfusp proteins were used in gel mobility-shift assays to test if Cfusp cDNA encodes a functionalprotein that can heterodimerize with CfEcR and bind tohsp27 EcRE. Neither CfEcR nor CfUSP alone retardedthe mobility of labeled EcRE (Fig. 9). When bothproteins were present, the mobility of the labeled EcREwas retarded. When excess unlabeled EcRE (25-foldand 200-fold) was used in the binding reaction, nobandshift was observed, whereas the use of 25-foldexcess of RORE, a nonspecific competitor, did not affectthis bandshift. However, some decrease in bandshiftoccurred with a 200-fold excess of RORE. Thus, CfUSPin the presence of CfEcR specifically bound to hsp27EcRE, indicating that the Cfusp cDNA encodes a func-tional protein.

DISCUSSION

Confirmation of Cloned cDNA as C. fumiferanaultraspiracle

We have cloned a cDNA from the spruce budworm, C.fumiferana, that showed high amino acid identity withthe usp proteins from D. melanogaster, B. mori, M.sexta, and A. aegypti. When translated in a TNTsystem, the Cfusp cDNA produced a 52-kDa protein.This size is similar to other insect usps. The deducedamino acid sequence of the longest ORF containeddomains characteristic of steroid/thyroid hormone recep-tor superfamily members. The overall Cfusp sequenceclosely resembled that of two other lepidopteran spe-cies, M. sexta [Jindra et al., 1997] and B. mori [Tzertzi-nis et al., 1994]. As expected, the C region was the mostconserved region in Cfusp. It contained the two cysteinezinc fingers required for the recognition and binding ofthe receptor to a hormone response element [reviewedby Freedman, 1992]. Although the A/B region showedthe least degree of conservation among all the usps,there was a stretch of 13 amino acids in Cfusp in theA/B region, that was 100% identical to all other insectusps.

In D. melanogaster, the functional ecdysone receptoris a heterodimer of EcR and usp proteins [Yao et al.,1992, 1993; Thomas et al., 1993]. Dmusp can also formheterodimers with mammalian thyroid and retinoicacid receptors [Khoury Christianson et al., 1992]. EcR/USP heterodimerization has also been shown in B. mori[Swevers et al., 1996] and A. aegypti [Kapitskaya et al.,1996]. BmEcR/BmUSP complex formation was stimu-lated in the presence of ecdysteroids [Swevers et al.,1996]. In A. aegypti, two usp isoforms have been

isolated, and both isoforms were able to form functionalheterodimers with AaEcR. Complex formation wasessential for DNA binding and hormone binding[Kapitskaya et al., 1996]. It was previously shown thatCfEcR can bind to EcRE in the presence of BmUSP[Kothapalli et al., 1995]. We have now shown thatCfEcR can form a complex with CfUSP. Both CfEcR andCfusp proteins were required for DNA binding (Fig. 9).The high amino acid identity with other usps, itsprotein size and its ability to bind to hsp27 EcRE inpartnership with CfEcR clearly demonstrated that thecDNA we cloned is C. fumiferana ultraspiracle.

Developmental Expression of Cfusp mRNA

C. fumiferana EcR-B mRNA is present throughoutlarval development with increases during ecdysteroidpeaks [Kothapalli et al., 1995]. Similarly, we observed a4.5-kb usp transcript in the embryos, in all larval stagesand in the pupae of C. fumiferana. Cfusp was expressedimmediately after oviposition but declined during em-bryonic development (Fig. 4). Whether the Cfusp mRNAwe detected during the early stages of embryonicdevelopment is newly synthesized in the embryo orcarried over maternal mRNA is unclear. Except in the6th instar larval midgut the Cfusp mRNA levels showedlittle variation during the larval and pupal stagestested (Figs. 4–8). The Cfusp mRNA levels increased inthe sixth-instar larval midgut but not in the epidermisand fatbody isolated from the same larvae. The CfuspmRNA levels did not show any change in the fifth-instar larval midgut. As reported earlier, CfEcR mRNAlevels increase in the sixth-instar larval midgut [Kotha-palli et al., 1995] as well as in the fifth-instar larvalmidgut during the ecdysteroid peaks [S.C. Perera, T.R.Ladd, and S.R. Palli, unpublished observations]. Thus,while EcR mRNA levels increase during all molts,Cfusp mRNA levels increase only in the sixth-instarlarval stage during the metamorphic molt. These obser-vations raise the possibility that there may be morethan one isoform of usp in the spruce budworm, andspecific isoforms may be required for interacting withEcR during larval and metamorphic molts. However,Northern hybridizations using total and isoform-specific probes showed that the expression of this uspisoform is similar to the total usp expression in themidgut. Therefore, if multiple Cfusp isoforms are pre-sent, the Cfusp that we have isolated is the majorisoform that is expressed in the midgut of the sixth-instar larvae. Another possibility is that only one usp ispresent in C. fumiferana. Multiple usp isoforms withdifferent N-termini, have been isolated in M. sexta[Jindra et al., 1997] and in A. aegypti [Kapitskaya et al.,1996]. The expression of mRNA for different Msuspisoforms is developmentally regulated. Msusp-1 mRNAwas expressed during intermolt periods, whereas theMsusp-2 mRNA showed increased expression duringthe larval and pupal molts. Jindra et al. [1997] sug-gested that Msusp isoforms 1 and 2 interact differen-

Choristoneura fumiferana usp 177

tially with the two M. sexta EcR isoforms. Whether theC. fumiferana EcR isoforms A and B have their ownheterodimeric usp partners remains to be seen.

In A. aegypti, the two usp isoforms are expressed in atissue specific manner. Aauspa and Aauspb isoformsare expressed mainly in the fat body and in the ovaryrespectively [Kapitskaya et al., 1996]. In C. fumiferana,we observed tissue-specific expression of usp in thesixth instar. In the epidermis and fat body, expressionof usp remained constant whereas in the midgut itincreased in the late larval stages (Fig. 7).

To date, only one usp isoform has been identified in D.melanogaster [Henrich et al., 1990] and in B. mori[Tzertzinis et al., 1994]. Dmusp mRNA was detected inall the developmental stages from embryo to adult [Oroet al., 1990, Henrich et al., 1994, Andres et al., 1993]. Asin the case of Cfusp, the highest levels of Dmuspexpression occur at the onset of metamorphosis [Hen-rich et al., 1994]. However, a direct correlation betweenthe increase in Dmusp expression, and ecdysone concen-trations in the hemolymph has not yet been estab-lished.

With the isolation and characterization of full-lengthcDNAs for Cfusp (this study), CfEcR [Kothapalli et al.,1995] and two ecdysone-induced transcription factors,CHR3 [Palli et al., 1996, 1997a] and CHR75 [Palli et al.,1997c], we have the necessary tools in hand to conductexperiments to assess the effects of over- and underex-pression of genes involved in ecdysteroid cascade oninsect development. We also plan to use Cfusp andCfEcR cDNAs for developing screening assays to iden-tify new stable ecdysteroids, as well as for elucidatingthe mode of action of the nonsteroidal ecdysteroidagonists RH-5992 and RH-2485.

ACKNOWLEDGMENTSThis study was supported by Canadian Forest Service/

Natural Sciences and Engineering Research CouncilResearch Partnership Program award 0192158, withthe Rohm and Haas Company.

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