Proc. Natl. Acad. Sci. USAVol. 90, pp. 3251-3255, April 1993Neurobiology

Precursor polyprotein for multiple neuropeptides secreted fromthe suboesophageal ganglion of the silkworm Bombyx modi:Characterization of the cDNA encoding the diapause hormoneprecursor and identification of additional peptides

(insect/peptide hormone/embryonic diapause/Phe-Xaa-Pro-Arg-Leu-NH2)

YUKIHIRO SATO*, MASAAKI OGUCHIt, NOBUO MENJOt, KUNIO IMAIt, HIROYUKI SAITOt, MOTOKO IKEDA§,MINORU ISOBE¶, AND OKITSUGU YAMASHITA§II*Radioisotope Research Center, Nagoya University, Chikusa, Nagoya 464-01, Japan; tLaboratory of Chemurgy, Faculty of Bioresources, Mie University, Tsu514, Japan; tAbrahi Laboratories, Shionogi and Co., Ltd., Koka, Shiga 520-34, Japan; §Laboratory of Sericultural Science and lLabomtory of OrganicChemistry, School of Agriculture, Nagoya University, Chikusa, Nagoya 464-01, Japan

Communicated by Fotis C. Kafatos, December 30, 1992 (received for review October 19, 1992)

ABSTRACT Peptidergic neurons, which serve as source ofvarious endocrine neuropeptides, were identified in the sub-oesophageal ganglion (SG) and brain of insects. In the silk-worm Bombyx mori, SG is known to secrete two neuropeptides,diapause hormone (DH) responsible for induction ofembryonicdiapause and pheromone biosynthesis-activating neuropeptide,which share a pentapeptide amide, Phe-Xaa-Pro-Arg-Leu-NH2(Xaa = Gly or Ser), at the C terminus. We have isolated cDNAclones for DH from the cDNA library of SG by using oligonu-cleotide probes. The molecular characterization of the cDNAreveals that the mRNA encodes an open reading frame con-sisting of 192 aa residues in which the 24-aa DH peptide islocalized at the N-terminal region just after the signal peptide.A homology search proposed that the cDNA encodes phero-mone biosynthesis-activating neuropeptide and three otherneuropeptides [a-, (3-, and y-SG neuropeptide (SGNP)] in theregion following DH, all of which are flanked by possibletryptic cleavage sites and share the Phe-Xaa-Pro-Arg-Leu-Glysequence at the C terminus. Northern hybridization analysisclearly showed that the gene expression was limited to SG. Wechemically synthesized a-, p-, and y-SGNP and used them toidentify components in extracts ofSG and to examine biologicalfunctions. a- and y-SGNP were identified in extracts of SG,and the synthetic P- and y-SGNP expressed weak DH activity.These results indicate that DH, along with four other neu-ropeptides, is generated from a common precursor polyproteinthat is encoded by a single mRNA transcribed in neurosecre-tory cells of SG.

In both vertebrates and invertebrates, the neuroendocrineaxis plays the central role in adaptation to changing environ-mental conditions through homeostatic and developmentalregulation. In insects, the neurosecretory system is deeplyinvolved in the regulation of molt, metamorphosis, repro-duction, and diapause, which are the critical events in theirlife cycle. Neurosecretory cells of the brain are well devel-oped to secrete several neuropeptides (1). Many peptidergicneurons are also localized in the suboesophageal, thoracic,and abdominal ganglia (2, 3). The suboesophageal ganglion(SG) was first identified in the silkworm Bombyx mori as aneuroendocrine organ that secretes diapause hormone (DH),which is responsible for induction of embryonic diapause(4-6). Recently, B. moriDH (Bom-DH) has been isolated andshown to be a 24-aa peptide amide (7, 8). Another function ofthe silkworm SG is to secrete the pheromone biosynthesis-

activating neuropeptide (PBAN), which stimulates the bio-synthesis of a sex pheromone, bombykol, in pheromoneglands of female moths (9-11). Two PBAN molecules, B.mori PBAN (Bom-PBAN) I and II, have been isolated fromadult heads of B. mori (9) and shown to consist of 33 and 34aa, respectively (10, 11). PBAN is now known to be identicalto the melanization and reddish coloration hormone, whichregulates coloration of integuments associated with phasepolymorphism ofthe armyworm Psedaletia separata (12, 13).The myotropic neuropeptides, myotropin and pyrokinin,which stimulate muscle contraction of hindguts and oviducts,have been isolated from the brain-SG complexes of thecockroach Leucophaea maderae (14, 15) and the locustLocusta migratria (16-18). DH, PBAN, myotropin, andpyrokinin differ from each other over the entire sequence butshare the conserved sequence, Phe-Xaa-Pro-Arg-Leu-NH2(FXPRLamide; Xaa = Gly, Ser, Thr, or Val) at their Ctermini, so these neuropeptides are classified as members ofthe FXPRLamide family (19).

It is generally accepted in vertebrates that multiple bioac-tive neuropeptides are released from a large common pre-cursor through posttranslational processing (20, 21). Thevariety of neuropeptides isolated from vertebrates has beenimmunocytochemically localized in insect neurons (2, 3),suggesting that insect neurosecretory cells also develop asimilar mechanism for bioactive neuropeptide production.The cytological observation localized only a pair of neuro-secretory cells in SG of B. mori (22), so DH and PBAN areexpected to be coexpressed in the same neurosecretory cell.However, it remains unknown whether these neuropeptidesare products of a common precursor polyprotein that isencoded by a single gene. We have recently cloned cDNAencoding DH by using oligonucleotide probes, which facili-tates the analysis ofthe mode of neuropeptide biosynthesis inSG of B. mori (8).

In the present paper, we describe the primary structure andexpression of the mRNA encoding a precursor peptide forDH.** The deduced amino acid sequence of the precursorsuggested that in addition to DH, PBAN and three additionalneuropeptides could be processed from the common precur-sor, which is translated from a single mRNA encoding DH.

Abbreviations: SG, suboesophageal ganglion; DH, diapause hor-mone; Bom-DH, Bombyx mori DH; PBAN, pheromone biosynthe-sis-activating neuropeptide; Bom-PBAN, B. mori PBAN; RP-HPLC, reverse-phase HPLC; SGNP, SG neuropeptide; Hez-PBAN,Helicoverpa (Heliothis) zea PBAN; FXPRLamide, Phe-Xaa-Pro-Arg-Leu-NH2 (Xaa = Gly, Ser, Thr, or Val).1"To whom reprint requests should be addressed.**The sequence reported in this paper has been deposited in theGenBank data base (accession no. D13437).

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This possibility was confirmed by chemical identification ofsome of these peptides from extracts of SGs. These resultsled us to conclude that some neurosecretory cells of insectshave developed a biosynthetic mechanism by which a set ofbioactive neuropeptides are generated from a common pre-cursor polyprotein through posttranslational processing.

MATERIALS AND METHODSAnimals. Commercial races of B. mori (Kinshu x Showa

and Asagiri) were used for experiments. The eggs wereincubated at 25°C under continuous illumination throughoutembryogenesis, and larvae were reared on fresh mulberryleaves or on artificial diets at 25-27°C under a 12-hr light/12-hr dark photocycle. Under these conditions, all of theemergent adults laid diapause eggs.

Screening of a cDNA Library. A cDNA library of SG-firstthoracic ganglion complexes of the Asagiri race was con-structed into Agt1O (23). A degenerate 35-mer [ACIGAIAT-GAAIGAIGAI(T/A)(C/G)IGAI(C/A)GIGGIGCICA] and anondegenerate 72-mer oligonucleotide (ACCGACATGAAG-GATGAGTCTGACCGTGGAGCCCACTCTGAGCGTG-GTGCTCTGTGCTTCGGCCCCCGCCTG), which were de-duced from the DH structure were synthesized (8), radiola-beled with [y32P]ATP by using T4 polynucleotide kinase, andthen used for screening by hybridization.DNA Sequencing. The DNA insert of positive clones was

amplified by PCR and directly sequenced by primer exten-sion using Taq polymerase (24). The sequence was edited andanalyzed by using DNASIS (Hitachi). The Swiss-Prot proteinsequence data base was searched for homologous sequences.

Hybridization Analysis. The PCR-amplified cDNA insert ofthe AcDH08 clone was labeled with [a-32P]dCTP (110 TBq/mmol; Amersham) by random priming (25). An actin cDNAcarrying a sequence from nt 729 to nt 949 of the actin A3 gene(26) was amplified from total RNA ofB. mori ovaries by PCRafter reverse transcription and then labeled at the 3' end with[a-32P]dCTP by using terminal deoxynucleotidyl transferase.

Total RNA was extracted from various tissues of pupaeand pharate adults by the method ofChomczynski and Sacchi(27). The isolated RNA was electrophoresed in an 0.8%agarose gel and then blotted onto a Hybond N+ membrane(Amersham). The hybridization was performed at 42°C in 6xSSC (lx SSC = 150 mM NaCl/15 mM sodium citrate)containing 50% formamide, 5x Denhardt's solution, 0.1%SDS, salmon sperm DNA (100 ,g/ml), and radioactiveprobes (1.5 x 105 Bq/ml) (28). The radiographic images werevisualized by a BAS2000 image analyzer (Fuji).

Synthetic Peptides. Peptides were synthesized as amidatedforms by using an ABI 431A peptide synthesizer (AppliedBiosystems) according to a fluoren-9-ylmethoxycarbonylprotocol and by a handmade peptide synthesizer employingbutoxycarbonyl chemistry (7, 8). Synthetic Bom-PBAN-I(10) was kindly provided by A. Suzuki (University ofTokyo).Bioassay ofDH activity was carried out by injecting samplesinto day-4 pharate adults of the N4 strain, and activity wasestimated by counting the number of diapause eggs afternonaffected (nondiapause) eggs have hatched, as described(refs. 6 and 7; see Table 1).

Isolation of Peptides. The synthetic peptide amides a-, 3,and y-)SG neuropeptides (SGNPs; see Results) were used asthe reference substances for extraction and purification. SGwith brain or with first thoracic ganglion was dissected fromabout 110,000 day-2 to day-3 pupae and used as the startingmaterial for isolation (8). The tissue homogenate was firstextracted by 80% ethanol as described (7, 8). Since thesynthetic SGNPs were recovered as 80% ethanol extracts,this fraction was dried, dissolved in 80Womethanol, and thenwashed with ether. The remaining solution was partitionedwith butanol. a- and t-SGNP were recovered in the butanol

extract, and 3-SGNP remained in the aqueous phase. Thebutanol extract was subjected to a reverse-phase HPLC(RP-HPLC) on a TSK gel octadecyl-4PW column (Tosoh,Tokyo). The column was eluted with a linear gradient of 0%oto 50% 2-propanol in 0.05% trifluoroacetic acid and thefractions corresponding to synthetic a- and y-SGNP werecollected separately. The peak material was rechromato-graphed for further purification. The isolated peak materialwas analyzed by RP-HPLC as described in Fig. 3 andsequenced using a PSQ-1 sequencer (Shimadzu).

RESULTSStructure of the Precursor Peptide Deduced from cDNA. By

using oligonucleotide probes, four clones encoding DH wereindependently isolated (8), and their inserts were sequenced.These four clones were identical to each other in the codingsequence but had various lengths of 5' and 3' noncodingregions. The nucleotide and deduced amino acid sequences ofAcDH08, which carried the longest insert (800 bp), are shownin Fig. 1. The open reading frame encoded a peptide con-sisting of 192 aa with a molecular mass of 22 kDa. A hydro-phobic sequence from Met-i to Cys-23 seemed to be a signal+1 CAACAACAAAA ATG TAT AAA ACC AAC ATT GTT TTC AAC 38+1 M Y K T N I V F N 9

GTT TTA GCT TTG GCA TTG TTC AGT ATT TTC TTC GCG 74V L A L A L F S I F F A 21

AGT TGC ACG GAT ATG AAG GAT GAA AGC GAC AGA GGA 110S C T D M K D E S D R G 33

DHGCT CAC AGT GAG CGG GGC GCT CTC TGG TTC GGC CCC 146A H S E R G A L W F G P 45

AGA CTC GGG AAG CGA TCA ATG AAG CCA TCC ACT GAA 182R L G K R S M K P S T E 57

GAT AAC AGG CAA ACC TTC CTG AGG CTG CTC GAG GCG 218D N R Q T F L R L L E A 69

GCT GAT GCC CTC AAA TTT TAC TAC GAC CAG CTA CCT 254A D A L K F Y Y D Q L P 81

TAC GAG AGG CAA GCC GAT GAA CCG GAA ACC AAA GTA 290Y E R 0 A D E P E T K V 93

ACA AAG AAG ATC ATC TTC ACC CCC AAA CTC GGG AGG 326T K K I I F T P K L G R 105

aAGC GTC GCC AAA CCC CAG ACG CAT GAA AGC CTC GAA 362S V A K P Q T H E S L E 117pTTCF

ATC CCC CGG CTC GGA AGGI P R L G R

CGG CTC TCT GAG GAC 398R L S E D 129PBAN

ATG CCT GCT ACG CCA GCT GAC CAG GAA ATG TAC CAA 434M P A T P A D 0 E M Y Q 141

CCT GAC CCC GAA GAA ATG GAG TCA AGA ACA AGA TAC 470P D P E E M E S R T R Y 153

TTC TCG CCC AGG CTG GGG CGC ACC ATG AGC TTT TCG 506F S P R L G R T M S F S 165

I

CCC AGA CTG GGA AGG GAG CTT TCG TAC GAT TAC CCT 542P R L G R E L S Y D Y P 177

ACA AAA TAT AGG GTT GCC AGA AGC GTT AAC AAG ACA 578T K Y R V A R S V N K T 189

ATG GAC AAC TAA ACGAATTATGGTCCGCTTGAGGTACCTCATT 621M D N 192

TGAGGTCTCGATCGACTCCGACGAACGGTTACGGGTAAACGGCGACA 668ATGTTAATGTTTTGGACGAAACAATTGTTAATTAATAAATTCACGTG 715ATTTTGGAATTGTAATTTATAAGTGAATAAAAAAATAAACTATTTAA 762AATAAATGAGTCATTATTATTATTAAAAAAAAAAAAAA - 800

FIG. 1. Nucleotide and deduced amino acid sequences of acDNA encoding DH. The sequences of Bom-DH, a-SGNP,3-SGNP, Bom-PBAN-II, and ySGNP are underlined.

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peptide (29). The sequence ofDH (Bom-[Trpt9]DH-I) (8) wasfound at aa 24-47. The DH sequence was followed by aminoacid residues, Gly-48, Lys-49, and Arg-50, which seemed tobe processing sites for the molecular maturation of DHthrough tryptic cleavage at Lys-49 and amidation of Leu-47by Gly-48, respectively (30).By homology search, we found that the cDNA encodes

Bom-PBANs (10, 11). Bom-PBAN-I was localized fromresidue 126 to residue 158 in the sequence, and Bom-PBAN-II was found from residue 125 to residue 158 (Fig. 1).Arg-125 was predicted to be the tryptic N-terminal cleavagesite for Bom-PBAN-I, and Arg-124 was predicted to be thesite for Bom-PBAN-II, indicating alternative N-terminal pro-cessing for molecular maturation of PBAN. Gly-Arg at res-idues 159 and 160 was predicted to provide the C-terminalprocessing site for proteolysis and amidation at the C termi-nus of both PBANs.

In addition to DH and PBAN, which have the conservedpentapeptide sequence FXPRL at their C termini, we foundFXPRL sequences at residues 118-122 and 164-168 and asimilar sequence, FXPKL, at residues 99-103. These threeFXPR/KL sequences were bounded by Lys-Lys or Gly-Arg,so that the following three peptides seem to be released fromthe precursor: (i) a heptapeptide localized at positions 97-103, designated a-SGNP; (ii) following a-SGNP, a heptade-capeptide (,B-SGNP) from residue 106 to residue 122; and (iii)an octapeptide (y-SGNP) from residue 161 to residue 168,which was localized adjacent to the C terminus ofPBAN. DHand a-SGNP were spaced with a sequence consisting of44 aaresidues bounded by dibasic amino acid residues. Followingthe C terminus of y-SGNP, there was a sequence consistingof 22 residues. Neither sequence had similarity to the knownpeptide sequences nor a conserved domain responsible forknown biological function. Thus, these two sequences are notbelieved to be processed into mature peptides. Conse-quently, the cloned cDNA is believed to encode the precur-sor polyprotein for DH, PBAN, and a-, P-, and y-SGNP.

Expression of the DH Precursor Gene. Accumulation ofmRNA in neural and nonneural tissues was examined byNorthern hybridization (Fig. 2). A hybridization signal wasobserved forRNA prepared from SG of day-0 pupae ofmixedsexes. The signal was detected as a single band of -900 bp,indicating that the characterized cDNA carried a nearlyfull-length cDNA insert. The brain-SG complex from day-4

C

A ( nOLm Xl) m m 0 L- L

B

-23S-16S

-16S

female pharate adults showed one positive band with anintensity comparable with that from SG from day-0 pupae. Inthe brain-SG complex of day-4 male pharate adults, the samesignal was also detected at an appreciable level. No positivesignal was found in other neural tissues such as brain aloneand thoracic and abdominal ganglia and nonneural tissuessuch as ovary, midgut, fat body, and trachea.

Identification of Predicted Neuropeptides and BiologicalActivity. The butanol extracts of SGs were separated intomany peaks by the first RP-HPLC step, and among them twopeaks corresponded to the synthetic a-SGNP and y-SGNP bytheir retention times (data not shown). The peak correspond-ing to a-SGNP was purified by the second RP-HPLC step. Onan analytical RP-HPLC, the purified material was eluted atthe same retention time (11.8 min) as that of the synthetica-SGNP (Fig. 3A). The amino acid sequence of the isolatedpeptide was Ile-Ile-Phe-Thr-Pro-Lys-Leu, which was identi-cal to a-SGNP. The peak corresponding to y-SGNP from thesecond RP-HPLC step was separated into two peaks by theanalytical RP-HPLC step: one peak was eluted at the sameretention time (18.6 min) as the synthetic y-SGNP, and theother peak was eluted at 8.1 min (Fig. 3B). The chromato-graphic behavior suggests that the peak at 18.6 min is naturaly-SGNP, although the peptide sequence has not yet beenanalyzed due to its small quantity. Interestingly, the 8.1-minpeak had the sequence Thr-Met-Ser-Phe-Ser-Pro-Arg-Leu,which is the same as that of y-SGNP, indicating that theunknown modification occurs during purification. A sub-stance showing a chromatographic behavior similar to that ofthe synthetic P-SGNP was found in the aqueous fraction butnot in the butanol fraction of ethanol extracts of SG; the finalidentification has not yet been completed.The five synthetic peptide amides, Bom-DH (Bom-[Trp19]-

DH-I; see ref. 8), Bom-PBAN-I (10), a-SGNP, 3-SGNP, andy-SGNP, were assayed for DH activity (Table 1). Bom-DHinduced diapause in 25% of the eggs when injected at a doseof 20 pmol per insect and gave a threshold amount of 1 pmolper insect. Bom-PBAN-I showed DH activity, but the rela-tive activity was 10 times lower and the threshold amountswere 100 times higher compared to Bom-DH. /3- and y-SGNPinduced diapause in 10% of the eggs at the maximum dosewith threshold amounts of 500 pmol per insect or more.a-SGNP showed no activity at any of the tested doses. Whenthese synthetic SGNPs were coinjected with Bom-DH at thesame molar ratios, there was neither a stimulatory nor aninhibitory effect of these peptides on DH activity (data notshown). Thus, a-, f3-, and y-SGNP have functions differentfrom that of DH.

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oFIG. 2. Expression of the gene encoding the DH precursor. (A)

Neural tissues [brain (Br), SG, a mixture of thoracic ganglia (TG),and a mixture of abdominal ganglia (AG)] and nonneural tissues[midgut (Mg), trachea (Tr), and fat body (Fb)] were collected fromday-0 pupae of both sexes for RNA extraction. Brain-SG (Br-SG)complexes and ovaries (Ov) were collected from male (M) and female(F) day-4 pharate adults. RNA (10-20 tg) was loaded on each laneand analyzed by Northern hybridization using cDNA (cDH08) as aprobe. 23S and 16S rRNA of Escherichia coli are indicated on theright as size markers. (B) The membrane was reprobed with an actincDNA probe after stripping off the cDHO8 probe.

A B

5 10 15 0 10 20Retention time (min)

FIG. 3. The analytical RP-HPLC profile of a-SGNP and y-SGNP.The purified a-SGNP (A) and y-.SGNP (B) from the second RP-HPLCstep were collected and analyzed on a TSK gel octadecyl-4PWcolumn with isocratic elution of 10%o and 7% 2-propanol in 0.05%trifluoroacetic acid, respectively (upper line). The synthetic a-SGNPand t-SGNP were analyzed under the same conditions (lower line).

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Table 1. DH activity of neuropeptides from the commonprecursor polyprotein

Relative activity,t Threshold amount,tNeuropeptide* % pmol per insect

Bom-DH 100 1a-SGNP,B-SGNP 500Bom-PBAN-I 10 130y-SGNP 1050

*All peptide amides were chemically synthesized and purified andinjected at various doses over two orders of magnitude into day-3.5pharate adults of the polyvoltine strain N4.

tActivity was measured by counting the number ofdiapause eggs outofthe total number offertilized eggs, and the dose required to inducediapause in 25% of the eggs (D25) was calculated from the dose-response (log[dose] vs. percentage of diapause eggs) curve. The D25of Bom-DH (Bom-[Trp19]DH-I; see ref. 8) was 5 pmol per insect.The relative activity was expressed as the reciprocal dose [(D25)-1]of the neuropeptide divided by that of Bom-DH times 100o.Relative activity was not estimated for a-, 3-, and -oSGNP, becausethey induced less than 25% diapause eggs. a-SGNP never induceddiapause eggs even at a dose of 20 nmol per insect.tThe threshold amount was determined as the dose at which thedose-response curve contacted the x axis.

DISCUSSIONIn the present paper, we have first demonstrated that theneurosecretory cells ofinsects generate functionally differentneuropeptides from a large common precursor encoded by a

single gene. The presence of the polyprotein precursor was

determined by sequencing the cDNA clone that was isolatedby using oligonucleotide probes for DH (8). From the struc-tural analysis of the precursor, we predicted that at least fiveamidated peptides, DH, a-SGNP, ,B-SGNP, PBAN, andy-SGNP, are released from the precursor through posttrans-lational processing (Fig. 1). The Northern blot analysisclearly showed that only SG contains a single species ofmRNA that hybridizes to the cDHO8 probe (Fig. 2). Further-more, the tissue-specific transcription of this mRNA isclosely correlated to the fact that only SG produces DH (6).Our preliminary experiments have indicated that the mRNAis translated into a polypeptide with a molecular mass of 20kDa in an in vitro system (unpublished data), suggesting thatthe common precursor is translated from a single mRNA. Theposttranslational processing of the polyprotein is confirmedby the chemical identification of DH, PBAN, a-SGNP, andy-tSGNP in extracts of SGs and adult heads (refs. 7 and 9; Fig.

3). Thus, we concluded that the neurosecretory cell of SGprovides the machinery for the limited cleavage of peptidesand the amidation of C-terminal amino acids.Most neuroactive peptides characterized thus far in mam-

mals are first synthesized as a large polyprotein from whichseveral peptides are cleaved (20, 21). The expression of thegene coding for the polyprotein can be regulated dependingon the cell or tissue type by splicing of a pre-mRNA andposttranslational processing of the polyproteins. In contrast,the SG secretory cell of B. mori exclusively transcribes a

single species of mRNA from the DH precursor gene andprovides a set of the multiple neuropeptides without anyalternative processing of prepropeptide.The mature neuropeptides may be released into circulation

and express their own function in the target organ as foundin DH and PBAN. Although the additional peptides share theFXPRLamide at the C terminus as DH and PBAN do, theywere far less active than DH in inducing diapause eggs in B.mori (Table 1). These synthetic peptides were recently as-

sayed for their pheromonotropic activity. 13-SGNP expressedhigherPBAN activity than Bom-PBAN-I, but a- and ySGNPwere far less active in pheromonotropic activity (unpublished

Aa

Bom 86 ADEPETKVT IFK R; HESTHLEFIPR R 124

1111 111111111 11111HiHez -31 TSSYSFTVTKKVIFTPKLGRSLAYDDKSFENVEFTPRLGR 9

PBAN -1 +1Bom 125 gLSEDMPATPADQEMYQPDPEEMESRTRYFSPRI R 160

Hez 10 RLSDDMPATPADQEMYRQDPEQIDSRTKYFSPRLGR 45

Bom 161 PMSFSPRI;RELSYDYPTKYRVARS 19211II111111111

Hez 46 TMNFSPRLGRELSYGKRLLTNLHNT 70

B

Bom-0 SVA-KPQTHESLEFIPRL11 III

He z-p SLAYDDKSFENVEFTPRL1111 1111111

Pss-PT KLSYDDKVFENVEFTPRL

FIG. 4. Sequence similarity of the Bom-DH precursor and theHez-PBAN precursor. (A) Alignment of the deduced amino acidsequence of the DH precursor and of the revised Hez-PBANprecursor (see text). Sequences corresponding to a-SGNP, ,-SGNP,y-tSGNP, and PBAN of B. mori are boxed. (B) Sequence similarityof ,B-SGNP of B. mori (Bom-,8), the corresponding peptide in theHez-PBAN precursor (Hez-,B), and pheromonotropin of P. separata(Pss-PT; see ref. 32).

data). The surgical extirpation of SG from B. mori pupaecauses adult wing deformation and inhibition ofoviposition inaddition to inhibition of diapause egg induction and phero-mone production. These phenomena might be regulated bySGNPs through the neural and/or neurohormonal action.

Recently, a gene for PBAN of Helicoverpa zea (Hez-PBAN) has been isolated and sequenced, from which thegene is proposed to encode two additional small neuropep-tides with FXPRLamide in addition to one PBAN molecule(31). However, there is no direct evidence that these neu-ropeptides are processed from the common precursor. Daviset al. (31) in their paper assigned a GTG codon to Met-i fortranslation initiation in the Hez-PBAN precursor. Ifthis GTGcodon is translated into Val-1 instead of the initiator Met-1 asfound in many genes, the coding sequence is extended furtherupstream. The longer peptide can be predicted from thenucleotide sequence (Fig. 4), although another candidate fora translation initiation site was not found. The revised versionof the sequence permitted us to generalize the organization ofthe FXPRLamide family of neuropeptides in the gene of bothspecies (Fig. 4A). The DH-like sequence was not found in theknown sequence of the Hez-PBAN gene, but homologscorresponding to a-SGNP, PBAN, and y-SGNP resided in itwith the same arrangement. The Hez-PBAN gene did notshare the ,B-SGNP-like sequence between a-SGNP andPBAN but encoded a sequence that is highly similar topheromonotropin ofP. separata (Fig. 4B). Thus, such similarorganization suggests that both genes have arisen from acommon ancestor, although some parts are diverged, pre-sumably satisfying the functional requirements of each insectspecies for a unique adaptation, such as embryonic diapausein silkworms (6) and phase polymorphism in armyworms (13).

We thank Professor A. Suzuki, University of Tokyo, for kindlyproviding the synthetic Bom-PBAN-I. The present study was sup-ported by a Grant-in-Aid for Scientific Research (03404008) and a

Grant-in-Aid for Encouragement of Young Scientists from the Min-istry of Education, Science and Culture of Japan.

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