Molecular Cloning, Genomic Organization, and Expression of a B-Type (Cricket-Type) Allatostatin Preprohormone from Drosophila melanogaster

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Biochemical and Biophysical Research Communications 281, 544–550 (2001)

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olecular Cloning, Genomic Organization, andxpression of a B-Type (Cricket-Type) Allatostatinreprohormone from Drosophila melanogaster

ichael Williamson,* Camilla Lenz,* M. E. Winther,†ick R. Nassel,† and Cornelis J. P. Grimmelikhuijzen*,1

Department of Cell Biology, Zoological Institute, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen,enmark; and †Department of Zoology, Stockholm University, SE-10691, Stockholm, Sweden

eceived January 23, 2001

Insects are a large group of animals that playcpedcnitcGie

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The insect allatostatins obtained their names becausehey block the biosynthesis of juvenile hormone (a ter-enoid) in the corpora allata (two endocrine organsear the insect brain). Chemically, the allatostatins cane subdivided into three different peptide groups: the-type allatostatins, first discovered in cockroaches,hich have the C-terminal sequence Y/FXFGLamide in

ommon; the B-type allatostatins, first discovered inrickets, which all have the C-terminal sequence(X)6Wamide; and the C-type allatostatins, first discov-

red in the moth Manduca sexta, which have an unre-ated and nonamidated C terminus. We have previouslyeported the structure of an A-type allatostatin prepro-ormone from the fruitfly Drosophila melanogaster.ere we describe the molecular cloning of a B-typerepro-allatostatin from Drosophila (DAP-B). DAP-B is11 amino acid residues long and contains one copy eachf the following putative allatostatins: AWQSLQSSW-mide (drostatin-B1), AWKSMNVAWamide (drostatin-2), <EAQGWNKFRGAWamide (drostatin-B3), EPTWN-LKGMWamide (drostatin-B4), and DQWQKLHGGW-mide (drostatin-B5). All five drostatins are noveleptide structures. The DAP-B gene has one intron andwo exons and is located at position 74B1 on the left armf the third chromosome. The gene is expressed in allevelopmental stages, but weakly in embryos andtrongly in larvae. In situ hybridizations of larvaehowed that neurons in the brain and abdominal gan-lia and endocrine cells in the gut expressed DAP-B.his is the first published report of a B-type allatostatinreprohormone in insects, and the first paper describinghe presence of B-type allatostatins in a representativef the insect order Diptera (flies). © 2001 Academic Press

The nucleotide sequence reported in this paper has been submittedo the GenBank Data Bank with Accession No. AF312379.

1 To whom correspondence should be addressed. Fax: 145-5321200. E-mail: [email protected].

544006-291X/01 $35.00opyright © 2001 by Academic Pressll rights of reproduction in any form reserved.

rucial roles in ecology and agriculture (e.g., in plantollination). Despite the importance of insects, how-ver, their molecular endocrinology is not well un-erstood. This will certainly change after the recentompletion and publication of the “Drosophila Ge-ome Project” (1–3). Because of these achievements

n Drosophila research, one can now anticipate thathe major future advances in insect molecular endo-rinology will occur by mining of the “Drosophilaenome Project” database and subsequent cloning of

mportant proteins that play central roles in thendocrinology of insects (4 – 8).The insect allatostatins are an important group of

nsect neurohormones that obtained their names,ecause they block the biosynthesis of juvenile hor-one (a terpenoid) in the insect corpora allata (two

ndocrine organs located near the insect brain). Ju-enile hormone is crucial for development (e.g., forhe maintenance of a larval stage) and reproductione.g., for oocyte formation). Chemically, the allatost-tins can be subdivided into three fully differenteptide groups: the A-type allatostatins, which wererst discovered in cockroaches (9, 10), and whichave the C-terminal sequence Y/FXFGLamide inommon; the B-type allatostatins, first discovered inrickets (11), which all have the C-terminal sequence(X)6Wamide; and a third group, first discovered in

he moth Manduca sexta (12) that we now call the-type allatostatins, which have a nonamidated C

erminus, and a structure unrelated to the A- and-type hormones. We have previously cloned the pre-rohormone for the A-type allatostatins from Dro-ophila melanogaster and, together with anotherroup, two potential receptors for these neurohor-ones (4 – 6, 13). In the present paper, we describe

he cloning of a B-type allatostatin preprohormonerom D. melanogaster.

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Vol. 281, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ATERIALS AND METHODS

Drosophila (Canton S strain) poly(A)1 RNA and cDNA were prepareds previously described (4, 7). Poly(A)1 RNA from embryos was pur-hased from Clontech. The initial oligonucleotide primers used in PCRere sense 59-CTAAGACGCGGAGAACTTACG-39 (corresponding toucleotide positions 11 to 31 of Fig. 2) and antisense 59-ACATGCCCTTCAGGTTGTTC-39 (corresponding to nucleotide posi-

ions 546 to 566 of Fig. 2), and the template was cDNA from 3rd instararvae. The PCR program used was 94°C for 3 min, then touchdownCR for 10 cycles, 95°C for 30 s, 65°C for 45 s, decreasing 1°C for eachycle and 68°C for 2 min, followed by 35 cycles of 95°C for 30 s, 55°C for5 s, 68°C for 2 min and a final extension of 68°C for 10 min. 39-RACEeactions were made with the above sense primer and the nested senserimer 59-CAGGGCTGGAACAAGTTCAGG-39 (corresponding to nucle-tide positions 496 to 516 of Fig. 2). 59-RACE reactions were made withhe above antisense primer and nested antisense primer 59-TTAGAGGGTGGACTGCCAGC-39 (corresponding to nucleotide posi-

ions 94 to 115 of Fig. 2). pCR4-TOPO (Invitrogen) was used for theloning. Sequencing reactions were carried out as described in thehermo Sequenase Radiolabeled Terminator Cycle Sequencing kit

Amersham). Northern blots were carried out as in (8), using a cDNArobe, corresponding to nucleotide positions 11-566 of Fig. 2. Probes forP49 were obtained as described in (14). In situ hybridizations were

arried out as in (15), using digoxigenin-labeled antisense riboprobes,orresponding to nucleotide positions 11–566 of Fig. 2, and stainingith alkaline phosphatase-conjugated anti-digoxigenin Fab fragments.olor development was with nitroblue tetrazolium.

ESULTS

Cloning of the Drosophila B-type prepro-allatostatinDNA. We screened the “Drosophila Genome Project”atabase with “electronic probes” corresponding to annprocessed B-type allatostatin sequence from theilkworm Bombyx mori, AWQDLNSAWG (16). This re-ulted in the alignment with a putative Drosophila

FIG. 1. Schematic representation of the Drosophila B-type allatosenomic clones. (A) Position of the cDNA clones. The lowest clone wasnd the upper clone of 59-RACE. (B) The cDNA of the B-type allatostave B-type allatostatins in black. (C) The allatostatin preprohormonehe exons are given as bars, being broad for the coding regions and sf the genomic BAC clone AE003524 from the “Drosophila Genome

545

ene (Gene CG6456; Accession No. AAF49354; locatedn a BAC clone with Accession No. AE003524), anno-ated by the “Drosophila Genome Project” as having annknown function. We designed primers against thexons of this putative gene, performed PCR, and sub-equently amplified a cDNA sequence (Fig. 1A), show-ng that the gene was actually expressed. After 39- and9-RACE, we finally obtained the complete cDNA se-uence (Figs. 1A and 1B).The cDNA contains a coding region of 633 nucleo-

ides, a 59-noncoding region of 282 nucleotides, and a9-noncoding region of 499 nucleotides, containing aoly(A)1 tail (Fig. 2). We did not find a polyadenylyla-ion signal in the 39-noncoding part. To be sure that theoly(A)1 stretch of 30 nucleotides was indeed theoly(A)1 tail, we carried out three separate 39-RACEeactions. They all ended at nucleotide position 1102 ofig. 2, suggesting that the poly(A)1 stretch was indeed

he tail.The cDNA of Fig. 2 codes for a protein of 211 amino

cid residues. We propose that the first ATG in thepen reading frame is the start codon, because it ishortly preceded by a stop codon (at nucleotide position9 to 27 of Fig. 2) and followed by a signal peptide

equence for RER membrane translocation, which isrobably cleaved off at Ala-28 (17).The prohormone contains the immature sequences of

ve B-type allatostatins that after their processing wouldave the following structures: AWQSLQSSWamide (dro-tatin-B1; at amino acid residue positions 66–75 of Fig.); AWKSMNVAWamide (drostatin-B2; at amino acidesidue positions 151–160 of Fig. 2); ,EAQGWNKFR-

in preprohormone cDNA, its gene, and the positions of the cDNA andoriginal PCR product, the middle clone was the product of 39-RACE,preprohormone. The signal sequence is highlighted in grey and the

ne. The intron is given as a line (not drawn to scale) and marked i1.l for the noncoding regions, and are marked E1 and E2. (D) Positionject”.

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AWamide (drostatin-B3; at amino acid residue posi-ions 164–176 of Fig. 2); EPTWNNLKGMWamide (dro-tatin-B4; at amino acid residue positions 179–190 ofig. 2); and DQWQKLHGGWamide (drostatin-B5; atmino acid residue positions 193–203 of Fig. 2). All fivemmature drostatin sequences are flanked by dibasic orribasic amino acid residues (KR or KRR), which arestablished endoproteolytic cleavage sites, and contain a-terminal Gly residue that is normally converted into a-terminal amide group (18). Furthermore, immaturerostatin-B3 contains an N-terminal Gln residue that isnown to be converted into a ,Glu (,E) group (19, 20).he five B-type drostatins are novel peptide sequenceshat have not been isolated, so far, from any other insectpecies (Table 1).

The Drosophila B-type prepro-allatostatin gene.omparison of the Drosophila allatostatin preprohor-one (DAP-B) cDNA with its corresponding genomic

equence, reveals the presence of one intron and twoxons (Figs. 1C and 2; Table 2). This comparison alsoeveals the presence of a small number of nucleotide

FIG. 2. The cDNA and deduced amino acid sequence of the Drohree clones (Fig. 1A). Nucleotides are numbered from 59- to 39-end, an the open reading frame. The position of intron 1 is indicated by ann grey. The translation termination codon is indicated by an asteriskines. The five immature B-type allatostatin sequences are underlinecid residues (KR or KRR) and contain a C-terminal Gly residue kn

546

ifferences between the two types of DNA, which, how-ver, does not lead to differences in amino acid residuesTable 3).

The “Drosophila Genome Project” has allocated theenomic BAC clone AE003524, which contains theAP-B gene (Fig. 1D), to position 74B1 on the left armf the third chromosome.

Comparison of DAP-B with other proteins. Therexist structural similarities between the five Dro-ophila B-type allatostatin sequences and the B-typellatostatins from crickets, stick insects, the protho-acicostatic peptide from the silkworm Bombyx mori,nd three myoinhibitory peptides from the locustocusta migratoria and the moth Manduca sexta

Table 4). But beside these similarities, there is noignificant structural similarity between DAP-B andhat of other proteins stored in the Swissprot orenBank databases. DAP-B, therefore, appears to be

he first published B-type allatostatin preprohor-one from insects.

hila B-type allatostatin preprohormone. The cDNA is composed ofamino acid residues are numbered starting with the first ATG codonrow and the exon nucleotides, bordering the intron, are highlighted-frame stop codons in the 59-noncoding region are underlined by thiny bold lines. These immature sequences are flanked by basic amino

n to be converted into a C-terminal amide group.

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Expression of the DAP-B gene. Northern blotshowed that the DAP-B gene is expressed in all devel-pmental stages, but mostly in larvae, and less inmbryos (Fig. 3). The size of the transcripts (1400 bp)orresponded well with our cloned cDNA (Figs. 2 and). In situ hybridization of 3rd instar larvae showedybridization signals in about 24 neuronal cell bodies.our pairs of cell bodies were in the protocerebral partf the brain (Figs. 4A, 4B, and 4E), and eight pairsere located in the ventro-lateral part of the abdomi-al ganglia (Fig. 4E). In the intestine, we found about0 cells in the midportion of the midgut that werexpressing the DAP-B gene (Fig. 4D). These cells havehe morphology of endocrine cells (Fig. 4B). Controls,sing sense probes showed no staining.

ISCUSSION

The insect A-type allatostatins are neurohormonesith pleiotropic functions. In some insects (e.g., cock-

oaches) they inhibit the biosynthesis of juvenile hor-one in the corpora allata, whereas in other insects

e.g., flies) this inhibitory effect is absent (21, 22). Inany (or perhaps all) insects, the A-type allatostatins

nhibit smooth muscle contractions of the gut or othernternal organs (21, 22). Also the B-type allatostatinsre multifunctional neurohormones. In crickets theynhibit (together with the A-type allatostatins) the bio-ynthesis of juvenile hormone in the corpora allata11), whereas in the locust Locusta migratoria, or the

oth Manduca sexta, this inhibition is confined to

B-Type Allatostatin Seq

Structure

Ala-Trp-Gln-Ser-Leu-Gln-Ser-Ser-Trp-NHAla-Trp-Lys-Ser-Met-Asn-Val-Ala-Trp-NH

,Glu-Ala-Gln-Gly-Trp-Asn-Lys-Phe-Arg-Gly-Ala-Trp-NHGlu-Pro-Thr-Trp-Asn-Asn-Leu-Lys-Gly-Met-Trp-NH

Asp-Gln-Trp-Gln-Lys-Leu-His-Gly-Gly-Trp-NH

Note. These are proposed sequences of mature B-type drostatins-terminal regions of the drostatins-B3 to -B5 (underlined) are still

TABLE 2

Intron/Exon Boundaries and Intron Sizeof the DAP-B Gene

Intron 59-Donor Intron size (bp) 39-Acceptor

1 CAG gtaagtg..... 3324 .....tccacag CTA

Note. Upper- and lowercase letters represent nucleotides in thexons and intron respectively. The sequence of the intron can beetrieved from the GenBank Data Bank, Accession No. AE003524.he position of the intron is shown in Figs. 1C and 2.

547

mooth muscle contraction (23, 24). Furthermore, a-type allatostatin from the silkworm Bombyx morias found to inhibit the biosynthesis of ecdysone (a

teroid hormone necessary for molting) in the silkwormrothoracic gland (16).We have recently cloned the preprohormone for four-type allatostatins from Drosophila (6). These A-typellatostatins are probably not acting on the corporallata in Drosophila, because immunocytochemicaltudies have shown that A-type allatostatins are ab-ent in nerve cells innervating these organs (25). Theloning of a preprohormone for the five B-type allatost-tins from Drosophila now offers the possibility that-type allatostatins are involved in the regulation of

uvenile hormone synthesis. Other obvious roles for the-type allatostatins from Drosophila are the regula-

ion of ecdysone biosynthesis in the Drosophila protho-

nces Present in DAP-B

Amino acid positions in Fig. 2 Name

2 66–75 drostatin-B12 151–160 drostatin-B22 164–176 drostatin-B32 179–190 drostatin-B42 193–203 drostatin-B5

e sequences of drostatin-B1 and -B2 are likely to be correct. Thecertain.

TABLE 3

Nucleotide Differences between the DAP-B cDNA of Fig. 2nd the Corresponding Genomic Sequences from the “Dro-ophila Genome Project”

Position ofthe nucleotidein the cDNA

Type ofnucleotidein the gene

Type ofnucleotide in

the cDNA

Change inamino acid

residue

286 G A —2176 T C —2194 A G —

399 A G Gln 3 Gln471 C G Val 3 Val941 T A —961 T C —988 T C —

1011 T C —1080 T A —1102 G C —

Note. The position of the nucleotide in the cDNA (Fig. 2) is given inhe first column, the type of nucleotide present in the genomic DNAn the second column, and the type of nucleotide present in the cDNAn the third column. The fourth column gives the amino acid residuencoded by the genomic (first row) and cDNA (second row) sequence.one of the nucleotide differences between the genomic DNA and

DNA leads to differences in amino acid residues.

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acic (ring) gland, and the control of smooth muscleontraction.

Using in situ hybridization, we found the presenceAP-B transcripts in endocrine cells of the midgut and

n different neurons of the CNS from 3rd instar Dro-ophila larvae (Fig. 4). These CNS neurons are clearly

Structures and Actions of

Amino acid sequence

A W Q S L Q S SWamide drosA W K S M N V AWamide dros

,E A Q G W N K F R G AWamide drosE P T W N N L K G MWamide dros

D Q W Q K L H G GWamide drosG W Q D L N G GWamide GrbG W R D L N G GWamide GrbA W R D L S G GWamide GrbA W E R F H G SWamide GrbA W D Q L R P GWamide GrbA W Q D L Q G GWamide CamA W Q D L N T GWamide CamG W Q D L Q S GWamide CamA W Q D L Q G AWamide CamA W Q D L Q A GWamide CamA W Q D L G S AWamide CamA W Q D L N S AWamide BomA W Q D L N S AWamide MasG W Q D L N S AWamide MasA W Q D L N A GWamide Lom

Note. The consensus sequence of B-type allatostatins is W(X)6Wam-terminal sequences of the drostatins-B3 to -B5 (underlined) are un

ther sequences are different.a The actions of the drostatins are unknown.b The Grb-AST-B1 to -B5 block juvenile hormone biosynthesis in tc The Cam-AST-B1 to -B6 block juvenile hormone biosynthesis in th

30).d Bom-PTSP blocks ecdysone biosynthesis in the prothoracic glande Mas-MIP1 and 2 are myoinhibitory in Manduca (24).f Lom-MIP is myoinhibitory in Locusta (23).

FIG. 3. Northern blots of different developmental stages fromrosophila. Each vertical lane contained 2.5 mg mRNA from embryos

0–24h); third instar larvae; pupae; and pooled adult male andemale flies. (A) Hybridization with a cDNA probe corresponding toucleotide positions 11–566 of Fig. 2. The number at the right giveshe size in kbp. (B) The blots from A were stripped and subsequentlyybridized with a cDNA probe corresponding to ribosomal protein 49RP49). This panel gives the loading efficiency.

548

istinct from the CNS neurons that have previouslyeen stained in Drosophila larvae, using an antiserumo A-type allatostatins (25). Because in situ hybridiza-ion only visualizes mRNA and, thereby, only neuronalell bodies, we do not know whether some of the labeledNS neurons (Figs. 4A, 4B, and E) project to the cor-ora allata and, thus, might be involved in the regula-ion of juvenile hormone biosynthesis. The presence ofAP-B transcripts in midgut endocrine cells (Figs. 4Cnd D), on the other hand, clearly suggests a regula-ory (inhibitory) role of Drosophila B-type allatostatinsn gut motility. Drosophila B-type allatostatins, there-ore, are brain-gut peptides, similar to Drosophila-type allatostatins (25) and tachykinin-related pep-

ides (15). The endocrine localization of B-type allatost-tins has not been described previously for any insect.All B-type allatostatins isolated and sequenced, so

ar, from insect species other than Drosophila have arp residue in the second and a Trp-amide residue inhe last position, and are nonapeptides (Table 4).ased on the presence of dibasic (KR) or tribasic (KRR)esidues in DAP-B (Fig. 2), we have now proposed thetructures of five Drosophila B-type allatostatins (Ta-les 1 and 4). Two of these allatostatins are nonapep-

own B-type Allatostatins

e Species

in-B1 Drosophila melanogaster (fruitfly)a





T-B1 Gryllus bimaculatus (cricket)b





ST-B1 Carausius morosus (stick insect)c






SP Bombyx mori (silkworm)d

IP1 Manduca sexta (moth)e

IP2 Manduca sexta (moth)e

IP Locusta migratoria (locust)f

e. W and Wamide of these sequences are highlighted by boxes. Thetain. The sequences of Bom-PTSP and Mas-MIP1 1 are identical, all

corpora allata of Gryllus (11, 29).orpora allata of Gryllus, but their actions in Carausius are unknown

Bombyx (16).

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ides, whereas three allatostatins appear to be-terminally elongated. If the neuroendocrine cellsroducing DAP-B also would produce a dipeptidyl ami-opeptidase, which processes neuropeptide intermedi-tes and removes N-terminally located X-Pro or X-Alaequences (26), then one peptide (drostatin-B4) wouldecome a nonapeptide with the structure TWNNLKG-Wamide, and another peptide (drostatin-B3) would

ecome a decapeptide starting with a protective ,Gluroup, ,EGWNKFRGAWamide (Table 4). Similarly, ifhe DAP-B producing neuroendocrine cells also wouldxpress a protease specific for acidic residues, which inome other animal groups is known to process neu-opeptide preprohormones (27, 28), drostatin-B5 wouldave the structure ,EWQKLHGGWamide. Thus, theroposed structures of the drostatin-B1 and -B2 areikely to be the correct final nonapeptide structures,hereas the sequences of the drostatins-B3 to -B5 have

till to be confirmed.Although the first insect B-type allatostatins have

een reported between five to nine years ago (11, 23,4), their preprohormones have not been published, soar. Our report on DAP-B, therefore, appears to be therst publication of an insect B-type allatostatin prepro-ormone. Furthermore, our report describes for therst time the presence of B-type allatostatins in aepresentative of the insect order Diptera (flies). Fi-ally, it also describes for the first time the localizationf B-type allatostatins in gut endocrine cells.

CKNOWLEDGMENTS

We thank Lotte Steffensen for typing the manuscript and Lund-eck Foundation, the Danish Biotechnological Research and Devel-

FIG. 4. Whole-mount in situ hybridizations of brain and gut frponding to nucleotide positions 11–566 of Fig. 2. The brains are viehe DAP-B gene (scale, 100 mm). (B) Larger magnification of the samhe midgut, containing endocrine cells expressing the DAP-B gene (uclei are unlabeled (scale, 50 mm). (E) Drawing, showing the positio

ocated in the protocerebrum (Pr) of the brain, whereas eight pairs

549

pment Program of the Danish Research Agency, Novo Nordiskoundation, and Director Ib Hendriksen Foundation for financialupport.

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