# Insect immunity. Characterization of a Drosophila cDNA encoding a novel member of the dipticin family of immune peptides

Claude Wicker, Jean-Marc Reichhart, Danièle Hoffmann, Dan Hultmark,
Christos Samakovlis, Jules Hoffmann

► To cite this version:

Claude Wicker, Jean-Marc Reichhart, Danièle Hoffmann, Dan Hultmark, Christos Samakovlis, et al. Insect immunity. Characterization of a Drosophila cDNA encoding a novel member of the dipticin family of immune peptides. Journal of Biological Chemistry, 1990, 265 (36), pp.22493-22498. 10.1016/S0021-9258(18)45732-8. hal-05256007

HAL Id: hal-05256007

https://univoak.hal.science/hal-05256007v1

Submitted on 15 Sep 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Insect Immunity

CHARACTERIZATION OF A *DROSOPHILA* cDNA ENCODING A NOVEL MEMBER OF THE DIPTERICIN FAMILY OF IMMUNE PEPTIDES*

(Received for publication, June 29, 1990)

Claude Wicker‡, Jean-Marc Reichhart‡, Danièle Hoffmann‡§, Dan Hultmark¶, Christos Samakovlis¶, and Jules A. Hoffmann‡

From the ‡Laboratoire de Biologie Générale de l'Université Louis Pasteur and Unité de Recherche Associée au Centre National de la Recherche Scientifique "Endocrinologie et Immunologie des Insectes," 12 rue de l'Université, 67000 Strasbourg, France and the ¶Department of Microbiology, University of Stockholm, S-10691 Stockholm, Sweden

*Drosophila* shows an immune response when challenged by injection of low doses of bacteria. To date, the molecules involved in this immune reaction have remained elusive, with the exception of cecropins (4-kDa antibacterial peptides initially isolated from the moth *Hyalophora cecropia*) for which three closely related genes have been characterized recently. We report the molecular cloning and sequencing of a cDNA from a library of immune *Drosophila* which encodes a novel member of the family of dipticerins (9-kDa antibacterial peptides initially isolated from the fly *Phormia terranovae*). Transcripts for the *Drosophila* diptericin are detected 2 h after injection of bacteria. They are apparently derived from a single gene mapping at position 56 A on the right arm of the second chromosome.

We discuss the existence of a distant relationship between the dipticerins and two other groups of antibacterial insect proteins, the attacins, and the sarco-toxins II.

Insect immunity is a rapidly expanding field of research. It is now well documented that members of the higher insect order Lepidoptera, Diptera, and Hymenoptera respond to septic or even aseptic injuries by the rapid synthesis of potent antibacterial peptides. These molecules, together with the long-known cellular reactions of phagocytosis and encapsulation, account for the remarkable resistance which these insects exhibit against numerous types of bacterial challenges. In recent years, several families of bactericidal peptides have been isolated from immune insects and their primary structures have been determined: cecropins (4 kDa) (1), attacins (>20 kDa) (2), dipticerins (9 kDa) (3), insect defensins (4 kDa) (4), and apidaecins (2 kDa) (5) (and various homologues referred to as sarcotoxins and sapecins (6-8)). These bactericidal molecules are mostly cationic peptides with a broad spectrum of activity against Gram-positive and/or Gram negative bacteria.

The predominant immune peptides in larvae of our laboratory insect *Phormia terranovae* (Diptera) are the anti-Gram-negative dipticerins (three closely related isoforms (3))

and the anti-Gram-positive insect defensins (two isoforms (4)). *In situ* hybridization studies indicate that mRNAs encoding both peptide families appear in the same blood cells and adipocytes within 1 h after injury of the larvae (9). A challenging problem regards the control of the concomitant transcription of the genes coding for these immune peptides. While large insects, like *Phormia*, *Sarcophaga*, and *Hyalophora*, have proven extremely useful for biochemical studies on immune peptides, their genome sizes and the poor genetic background are relatively unfavorable for studies on the regulation of the transcription of these genes.

*Drosophila*, a dipteran distantly related to *P. terranovae*, obviously represents a better experimental model for such studies. Previous studies on the immune response in *Drosophila* (10-14) suggested the existence of many similarities with that of other Diptera and Lepidoptera, and the genes coding for a *Drosophila* homologue of cecropins, immune peptides that were initially isolated from the moth *Hyalophora cecropia* (1), have been recently characterized (15).

In this paper, we have addressed the question as to whether immunized *Drosophila* produce peptides structurally related to the dipticerins of *P. terranovae*. We report the molecular cloning of a *Drosophila* cDNA encoding a peptide with significant homology to *Phormia* dipticerins. We show that transcription of *Drosophila* diptericin occurs rapidly after injection of bacteria.

## MATERIALS AND METHODS

*Insects, Immunization, and Antibacterial Assay*—Experiments were performed with the Canton S wild-type strain of *Drosophila melanogaster*, grown on standard medium at 20 °C.

Adult insects were immunized by injection of 0.1-0.2 µl of a diluted log-phase culture of *Escherichia coli* (500 live bacteria) and were returned to feeding tubes. One day after injection, groups of 20 flies were homogenized in 10% acetic acid to remove the larger proteins and the proteases from the medium. The homogenate was centrifuged for 5 min at 250 × g. Acetic acid extraction was repeated on the pellet, and the resulting supernatants were pooled. The acetic acid from the supernatant was removed by rotary vacuum evaporation. The residue was resuspended in 8 µl of distilled water and its activity tested on 2-µl aliquots against *E. coli* D31 and *Micrococcus luteus*. Antibacterial activities were recorded as growth inhibition produced in thin agar plates seeded with each of these strains, as previously described (2, 16).

*Cloning Procedure*—We screened 30,000 pfu (10,000 pfu/13-cm Petri dish) of a cDNA library constructed in the vector λZAP II using polyadenylated RNA extracted from adult males 18 h after immunization (15). The probe was a pool of synthetic oligomers (5' GT CCA G/CAC T/CTT T/CTG G/ATG 3'), complementary to residues 38-43 of diptericin A from *Phormia* (3), end-labeled with [γ-³²P]ATP and purified on a gel-filtration column (AcA 202, LKB). Plaque lifts on nitrocellulose filters (BA 85, 0.45 µm, Schleicher & Schüll) were

<footnote>* This research was supported by the Swedish Research Council exchange programme, by the Swedish Natural Science Research Council, and by a Carl Tryggers Stiftelse för Vetenskaplig Forskning. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.</footnote>

<footnote>§ To whom correspondence should be addressed.</footnote>
performed as described (17). After baking for 2 h at 80 °C under vacuum, the filters were prehybridized at 42 °C for 3 h in 5 × SSC (standard sodium citrate), 50 mM phosphate buffer, pH 6.5, 5 × Denhardt's solution, 100 µg/ml denatured salmon sperm DNA, 0.1% sodium dodecyl sulfate. Hybridization was performed in the same solution at 42 °C overnight. The filters were washed three times for 15 min at room temperature in 2 × SSC, 0.1% sodium dodecyl sulfate and subjected to autoradiography.

**Sequencing of DNA**—DNA sequences were determined by the dideoxy-oligonucleotide chain-termination method (18) on double-stranded bluescript DNA and on single-stranded DNA, after subcloning the insert into M13mp19 phage (19). The products were separated on polyacrylamide gels (8% acrylamide, 7 M urea).

**DNA and RNA Analysis**—DNA extraction from adult flies, Southern blots of restriction endonuclease-treated DNA, hybridization at high stringency, and washing of the filters all followed standard procedures (20).

Total RNA, extracted by the lithium chloride/urea procedure (21) was separated by electrophoresis on denaturing formaldehyde agarose gels in MOPS<fnref n="1" /> buffer (22). Northern transfers were performed as described (23). Prehybridization and hybridization of the filters with the diptericin clone from *Drosophila* (Figs. 3–5) was at high stringency as described (20).

For *in situ* hybridization, polytene chromosome squashes were hybridized with 10⁶ cpm of ³⁵S of the random primed cDNA probe and exposed for 72 h.

For RNase protection assay, the flies were immunized and after 6 h RNA was extracted as described (15). For the probe, the Bluescript plasmid with the diptericin cDNA insert was linearized with NdeI. Transcription from the T7 promoter was done as described (24). The probe protected 186 nucleotides at the 3' end of the transcript. Hybridization was overnight at 45 °C. RNase digestion was done at 30 °C for 45 min (24). The protected fragments were loaded on a 6% sequencing gel together with sequencing ladders as markers.

## RESULTS

**Antibacterial Activities in Normal and Immune Drosophila**—We first confirmed that in our conditions *Drosophila* responds to a bacterial challenge by building up an immune response. We injected third instar larvae and adults with a low dose of bacteria and monitored the presence of an antibacterial activity after 24 h in whole body extracts. The activity was measured by the growth inhibition assay on agar plates seeded either with the gram negative *E. coli* D 31 or with the Gram-positive *M. luteus*. As is apparent from Table I, both larvae and adults showed a significant antibacterial activity against the Gram-negative and the Gram-positive test organisms. Similar assays performed on normal insects revealed no constitutive antibacterial activity in larvae or adults. However, a marked activity was observed in normal early pupae (day 1–2) and was directed against both types of bacteria. In contrast, late (day 4–5) pupae were devoid of any activity. Bacterial challenges performed in pupae yielded inconclusive results because of a high rate of mortality.

**RNA Hybridization**—Assuming that the anti-Gram-negative activity monitored in challenged adults might result in part from the synthesis of a diptericin homologue, we have designed a pool of oligodeoxyribonucleotide probes (17-mer, 16 possibilities) complementary to residues 38–43 of diptericin A from *Phormia*, using the preferential codon choice of *Drosophila* (25). The probe pool (5' GT CCA G/CAC T/CTT T/ CTG G/ATG 3') was end-labeled with γ-³²P and hybridized to total RNA from normal and immune (24-h post-inoculation of bacteria) adults of *Drosophila*. This probe gave a strong signal with RNA from immune insects but not with RNA from normal insects (data not shown). The major band was at ~0.7 kilobase(s); two fainter signals were occasionally observed in immune RNA at 2.5 and 9.5 kilobase(s).

<caption>TABLE I</caption>

Antibacterial activities in extracts of *Drosophila*

For each assay, 20 insects were subjected to whole body extraction in acetic acid and after dessication, the residue was resuspended in 8 µl of distilled water. Aliquots of 2 µl were tested in the growth inhibition assay. Results are given as +, clearly detectable inhibition and –, no detectable inhibition. This experiment was repeated several times yielding similar results.

<table><thead><tr><th rowspan="2"></th><th colspan="2">Bacterial strains</th></tr><tr><th><i>E. coli</i> D 31</th><th><i>M. luteus</i></th></tr></thead><tbody><tr><td colspan="3"><strong>Control insects</strong></td></tr><tr><td>Larvae (3rd instar)</td><td>0</td><td>0</td></tr><tr><td>Pupae (early, 1–2 days)</td><td>+</td><td>+</td></tr><tr><td>(late, 4–5 days)</td><td>0</td><td>0</td></tr><tr><td>Adults (4 h)</td><td>0</td><td>0</td></tr><tr><td>(2 days)</td><td>0</td><td>0</td></tr><tr><td>(7 days)</td><td>0</td><td>0</td></tr><tr><td colspan="3"><strong>Experimental insects<sup>a</sup></strong></td></tr><tr><td>Larvae (3rd instar)</td><td>+</td><td>+</td></tr><tr><td>Adults (2 days)</td><td>+</td><td>+</td></tr></tbody></table>

<sup>a</sup> Insects were challenged with an injection of ~500 log-phase cells of *E. coli*, 24 h before extaction as in 1.

<figure><img src="image_2.png" /><figcaption>**FIG. 1. Nucleotide sequence of a cDNA encoding *Drosophila* prediptericin.** The clone consists of 466 nucleotides and shows an open reading frame, beginning with an ATG triplet, which allows us to deduce a 106-residue peptide as shown below the nucleotide sequence. The sequence complementary to the probe in the diptericin coding region is also *underlined*. The pentaglycine region is *boxed*. Note the presence of a *Hind*III restriction site (AAGCTT) beginning at the second nucleotide of the stop codon.</figcaption></figure>

**Isolation of cDNA Clones**—The radioactive probe pool was used to screen a cDNA library which had been prepared from immune adults (18-h post inoculation of bacteria) of *Drosophila* (15) and 9 hybridization-positive clones were isolated which contained cDNA inserts between approximately 0.1 and 0.5 kilobase pairs. We have focused our attention on the clone containing the largest insert. The sequence of this insert is shown in Fig. 1 and consists of 466 nucleotides. The largest open reading frame beginning with an ATG triplet allows us to deduce a 106-amino acid peptide. The comparison of the deduced peptide sequence with that of diptericin A from *Phormia* (Fig. 2) shows extensive homologies leaving no doubt

<footnote n="1">¹ The abbreviation used is: MOPS, 4-morpholinepropanesulfonic acid.</footnote>
**SIGNAL PEPTIDES:** Suggested cleavage

Dros. Dipticerin -23 MQ-FTIAVALLCCAIASTL--A VPM P -1

Phorm. Dipticerin -18 MKLFY---LLUICALS-L--A$^*$U-MA$^{(4)}$-1

Sarcotoxin II -24 MKSFUFFAACMAIIALSSLUQA$^*$YP -1

**P DOMAIN:**

Dros. Dipticerin 1 DD--MTMKPTPPPQYPL 15

Phorm. Dipticerin 1 DEKPKLILPTAPPNLP 17

Sarcotoxin II 1 Q*--KLPUPIPPPTNPP 14

**G-LIKE DOMAINS:**

Sarcotoxin II, G₁ 137 ORLGASISRDUARGUS-DTLTKSUSANLFRNDN--HNLDASU-E--R-SDURQNHGFNFQKTGGMLDYS--HA 200

Attacin, G₁ 57 NRGHLGLSLTDIHI PGFG-DKMIAAGKUNVEHNDN--HDITAKA-FATR-NMPDIANUPNFNTUGGGIDYM-EK 122

Dros. Dipticerin 16 NLQGGGGG-QSGDGFG---FAUQHGKUWTSDNGRHEGLNGGYGQHLGGPYGNSEPSWK-UGSTYTYR-E-PNF 83

Phorm. Dipticerin 18 QLUGGGGG-NRKDGFG---USUDAHQKUWTSDNGRHSIGUTPGVSYQHLGGPYGNSRPDYR-IGAGVSYN-E-G* 83

Sarcotoxin II, G₂ 201 NRGHLNAGLIRFSGIG-NQATUGGYSTLERSNGLTSLKANAGGSQULSGPFANQR-DYS-FGLGLSHNAURG* 270

Attacin, G₂ 123 DKIGASAAAHQDFINRNDYSLDGKLNLEKTPD--TSIDFNAGFKF-DTPFMKSSWEPH-FGFSLSKY-E 188

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>FIG. 2. Sequence comparison between the amino acid sequences of *Drosophila dipticerin* (deduced from the cDNA sequence in Fig. 2), *Phormia dipticerin* D (cDNA clone D15) (26), and parts of *Sarcophaga sarcotoxin* IIA (7) and *Hyalophora acidic* attacin (27). Sequence identities with one or the other dipticerin are shown with bold typeface; identities between G₁ domains of sarcotoxin II or attacin and any of the other domains are underlined. Further identities exist between attacin and sarcotoxin II (7), but are not enhanced. Secondary amino acid modifications are indicated with asterisks: the NH₂-terminal residue in sarcotoxin II is pyroglutamate, and the COOH-terminal glycine residues in *Phormia* dipticerin and in sarcotoxin II are lost in the formation of COOH-terminal amide groups. The signal peptides are aligned with the most likely cleavage site for signal peptidase (29) in the same position; an alternative possible cleavage site is indicated for *Phormia* dipticerin. The inset illustrates the suggested domain structures of *Drosophila dipticerin*, *Sarcophaga* sarcotoxin, and *Hyalophora* attacin (see "Discussion"). The structure of attacin pre- and propeptides is from Ref. 28.</figcaption></figure>

that the *Drosophila* clone codes for a dipticerin homologue. In particular, the pentaglycine stretch characteristic of the *Phormia* dipticerin family is present in the *Drosophila* peptide in a similar position. Two regions of perfect homology extending over 10 and 12 amino acids are also evident as well as several minor stretches with identical sequence. A weak homology is also seen to the much larger antibacterial proteins attacin and sarcotoxin II (see "Discussion").

The sequence analysis of the mRNA of *Phormia* dipticerin had shown previously that this antibacterial peptide is synthesized as a prepeptide with a putative signal sequence of 18 predominantly hydrophobic amino acids which precedes the essentially hydrophilic NH₂-terminal amino acids of the mature peptide (26). The peptidic sequence deduced from the *Drosophila* clone shows a similar situation with 19-hydrophobic amino acid residues preceding a hydrophilic stretch of residues. Some degree of uncertainty remains as regards the exact cleavage site of the signal peptide in the *Drosophila* dipticerin homologue until the NH₂ terminus of the native mature peptide can be determined by amino acid sequencing. However, we tentatively assume that the cleavage removes the 23 first NH₂-terminal residues and that the mature peptide starts with Asp (as in *Phormia* dipticerin). If our assumption is correct, the mature dipticerin peptide of *Drosophila* consists of 83 residues and has a molecular mass of 8993 Da (*Phormia* dipticerin A, the only member of the dipticerin family which has been fully characterized has 82 residues and a molecular mass of 8611 Da).

The termination codon in the *Drosophila* dipticerin clone is followed by a 3'-non-translated sequence of 121 nucleotides. There is no consensus polyadenylation sequence (AATAAA) in the clone.

*Transcriptional Profiles for the Drosophila Dipticerin Homologue*—Transcriptional profiles were investigated on total RNA extracted from normal and experimental *Drosophila*

<figure><img src="image_5.png" /><figcaption>FIG. 3. RNA blot hybridization of *Drosophila dipticerin* cDNA. 20 µg of each RNA were analyzed by Northern blot hybridization under high stringency conditions by using the nick-translated insert of the dipticerin cDNA clone from *Drosophila* as a probe. Expression of the dipticerin gene in response to an injection of bacteria. RNA from male adults were harvested at different times after the injection of bacteria (0, 2, 4, 6, 18, and 24 h). kb, kilobase(s).</figcaption></figure>

using the dipticerin cDNA clone as a hybridization probe.

The cDNA hybridized under conditions of high stringency to one single band at ~0.7 kilobase. Assuming a poly(A) tail of 150-200 nucleotides, the dipticerin gene must encode a mRNA of at least 500-550 nucleotides, which is consistent with the length of the insert in our clone.

A time course study performed on adult males challenged by the inoculation of a low dose of bacteria showed that dipticerin transcripts were present as early as 2-h post-inoculation. The hybridization signal increased to its maximum at 4 h after which it became fainter (Fig. 3).

Transcription of dipticerin was investigated by RNase protection analysis throughout various developmental stages in control as well as in stimulated insects (Fig. 4). The results show that a marked response was induced in stimulated
<figure><img src="image_6.png" /></figure>

FIG. 4. Expression of the diptericin gene during development. RNA from untreated or vaccinated animals were assayed by RNase protection. Early and late third instar larvae refers to feeding and wandering stages, respectively; white, white prepupae; early pupae, up to 49 h after pupariation; late pupae, 49 h until eclosion. Ten µg of total RNA was used for each hybridization and the gel was exposed for 2 h.

<figure><img src="image_7.png" /></figure>

FIG. 5. Southern blot of BamHI (B1), EcoRI (E1), and HindIII (H3) digested genomic Drosophila DNA hybridized at high stringency with the diptericin cDNA probe. The molecular masses (in kb) of markers are indicated at the right of the figure. The presence of a HindIII restriction site in the diptericin cDNA (see Fig. 1) generates two bands hybridizing with the diptericin probe (lane H3).

insects regardless of the developmental stages investigated, with the exception of early pupae which responded only faintly. Interestingly, unchallenged early pupae and adults appeared to contain low but significant amounts of diptericin transcripts which were, however, not detected in embryos and larvae. In comparison to cecropin transcripts (15), the diptericin transcripts were found to be very abundant in induced insects. Equal loading and integrity of the RNA were tested in Northern blots probed with actin (data not shown).

*Existence of a Single Diptericin Gene and Cytological Localization—*High stringency hybridization of EcoRI or BamHI digested genomic DNA on Southern blots with the cDNA

<figure><img src="image_8.png" /></figure>

FIG. 6. Localization of the diptericin gene by *in situ* hybridization of the labeled cDNA probe with polytene chromosomes. The diptericin cDNA probe maps to the 56A region in the middle portion of the right arm of the second chromosome (indicated by the arrowhead).

insert probe gave a single signal. In the case of digestion with HindIII, two bands hybridized with the diptericin probe (Fig. 5), since a HindIII restriction site is present in the cDNA as apparent in Fig. 1. The chromosomal position of the diptericin locus was mapped by hybridization to salivary gland polytene chromosomes (Fig. 6). A single signal was observed at position 56 A, on the right arm of the second chromosome.

DISCUSSION

Our results demonstrate that *Drosophila* synthesizes a mRNA encoding a homologue of the dipticerins recently described in the Dipteran *P. terranovae*. The synthesis of this mRNA can be induced by the inoculation of bacteria, indicating that it is part of the immune response of this insect. Other molecules are obviously involved in this response in *Drosophila*, as the transcription of several cecropin genes has also been observed in similar conditions (15).

So far, no structural information has been published on dipticerins or diptericin-related molecules from insects others than *Phormia*. The homologies between *Phormia* and *Drosophila* dipticerins are particularly striking as regards the following criteria: (i) the molecular masses (~9 kDa) of the two peptides; (ii) the hydrophilicity profiles; (iii) the amino acid compositions. Both peptides are remarkably rich in Gly (18, 22%, respectively) and Pro (11%, 10%). This richness has undoubtedly a major influence on the tertiary structure of both peptides and hence on their mode of action. (iv) The primary sequence, which shows an overall homology of approximately 60%, with several regions (of 4 residues and more) of full homology between positions 19 and 69. As already noted, the characteristic pentaglycine stretch of the *Phormia* dipticerins is present in an equivalent relative position in *Drosophila*. It has been suggested that this sequence confers extreme flexibility to the peptidic chain (3).

Conceptually, the diptericin molecules can be divided in two major domains. At the NH₂-terminal there is a short, very proline-rich “P domain.” Five of the first 15 residues are Pro in *Drosophila*, 6 of 17 in *Phormia*. This remarkable richness in Pro residues is reminiscent of another family of antibacterial peptides, the apidaecins, that were recently isolated from honeybees (5). In these small (2.1 kDa) peptides, Pro accounts for 33% of the total amino acid content. The remaining part of the diptericin molecule constitutes a long Gly-rich “G domain,” where the pentaglycine stretch is situated near the junction to the P domain.

Interestingly, the *Drosophila* diptericin, like its *Phormia* counterparts, appears to be distantly related to two classes of much larger insect antibacterial peptides, the attacins and the attacin-related sarcotoxins II. Both classes of molecules have two G domain like sequences adjacent to the COOH-terminal (Fig. 2), although they are less Gly rich. The similarities in pairwise comparisons of the different G domains range between 11% (sarcotoxin II G₁-*Phormia* diptericin) and 34%
identity (sarcotoxin II G<sub>2</sub>-Phormia diptericin), and in most cases they are better than 20%. Many of the differences represent conservative amino acid replacements. Furthermore, a P domain can be identified at the NH<sub>2</sub>-terminal of sarcotoxin II, but not in the attacins. The absence of a P domain in the attacins may be related to the additional processing step that occurs in these proteins where an NH<sub>2</sub>-terminal propeptide is proteolytically removed (27). Further support for the suggested domain structure comes from Sun et al.,<fnref n="2" /> who recently found that each G domain in the attacin genes is encoded by a separate exon. The conservation of the P and G domains implies that they may correspond to functional units, and that the diptericin, attacin, and sarcotoxin II families are phylogenetically related. Thus the dipticerins, with just a P and a single G domain, appear to define the minimal member of a new superfamily of related bactericidal proteins.

By analogy with *Phormia*, we assume that the mature NH<sub>2</sub>-terminal of *Drosophila* diptericin is at the Asp(+1) residue (Fig. 2). However, the most likely site for signal peptidase cleavage is between Ala(-5) and Tyr(-4), as predicted by the algorhythm of von Heijne (29). The mature end of diptericin may then be created by the further removal of two successive X-Pro dipeptides by dipeptidyl peptidase, similar to the processing of the proforms of the inducible antibacterial peptides cecropins A and B in *Hyalophora cecropia* (30) and of many other small peptides where X-Pro or X-Ala dipeptides are trimmed off (31). A similar scheme has also been suggested for the processing of sarcotoxin IIA (7). The *Phormia* diptericin is more problematic, but in this case the signal peptidase may in fact cut directly at the NH<sub>2</sub>-terminal of the mature peptide. This position is the second most likely cleavage site (probability score 8.57 *versus* 9.38 for the position at Ala(-4), on a scale where most sites get negative scores).

For *Phormia* diptericin, peptide sequencing data indicated that the COOH-terminal residue is a Phe (3). Given that the mRNA coding for this peptide shows a triplet for Gly between the Phe triplet and the STOP codon, it was inferred that the mature *Phormia* diptericin is amidated (26). COOH-terminal amidation appears to be relatively common among the antibacterial peptides involved in the immune response of insects (cecropins (32, 33), sarcotoxin II (7)), and is also observed in other low molecular weight antibacterial peptides such as melittin (34). It has been demonstrated in the case of cecropins that COOH-terminal amidation enhances the antibacterial activity of the peptide (35, 36). In contrast, the present data suggest that the *Drosophila* diptericin is not amidated. Indeed, when the sequences for *Phormia* and *Drosophila* dipticerins are aligned as shown in Fig. 2, it is apparent that the *Drosophila* peptide has an additional COOH-terminal stretch of three amino acids, when compared to that of *Phormia*, and that the STOP codon is not preceded by a Gly, excluding the possible action of a peptidyl-glycine-α-amidating monooxygenase.

Our data on the hybridization of the *Drosophila* cDNA to genomic DNA and to polytene chromosomes from salivary glands indicate that transcription occurs from a single gene located in a relatively poorly investigated region of the second chromosome. This differs from the situation observed for cecropin in *Drosophila* for which a cluster of three active genes (plus two pseudogenes) at locus 99 E has been found (15).

Of great potential interest is the observation that constitutive expression of the diptericin gene occurs in early pupae

and in adults. A similar result has been observed for cecropins in *Drosophila* (37). Constitutive expression of another inducible antibacterial peptide, insect defensin, has also been observed in pupae of *Sarcophaga* (38) and *Phormia* (9). These results suggest that at least some of the inducible immune peptides of insects might play specific roles in normal development. The constitutive expression of the diptericin and cecropin genes in early pupae of *Drosophila* probably accounts for the strong antibacterial activity which we have monitored in whole extracts of such animals in this study (Table I).

We are now extending our studies to the control of the expression of the *Drosophila* diptericin gene in normal and challenged insects. The role(s) of diptericin in normal development will also be a center of interest.

*Acknowledgments*—We are indebted to Dr. Geoff Richards for advice in the *in situ* hybridization on polytene chromosomes, and for critical reading of the manuscript, and to Dr. Deborah Kimbrell for help with staging the animals.

REFERENCES

1. Steiner, H., Hultmark, D., Engström, A., Bennich, H., and Boman, H. G. (1981) *Nature* **292**, 246-248

2. Hultmark, D., Engström, A., Andersson, K., Steiner, H., Bennich, H., and Boman, H. G. (1983) *EMBO J.* **2**, 571-576

3. Dimarcq, J. L., Keppi, E., Dunbar, B., Lambert, J., Reichhart, J. M., Hoffmann, D., Rankine, S. M., Fothergill, J. E., and Hoffmann, J. A. (1988) *Eur. J. Biochem.* **171**, 17-22

4. Lambert, J., Keppi, E., Dimarcq, J. L., Wicker, C., Reichhart, J. M., Dunbar, B., Lepage, P., van Dorsselaer, A., Hoffmann, J. A., Fothergill, J., and Hoffmann, D. (1989) *Proc. Natl. Acad. Sci. U. S. A.* **86**, 262-266

5. Casteels, P., Ampe, C., Jacobs, F., Vaeck, M., and Tempst, P. (1989) *EMBO J.* **8**, 2387-2391

6. Okada, M., and Natori, S. (1985) *J. Biol. Chem.*, **260**, 7174-7177

7. Ando, K., and Natori, S. (1988) *Biochemistry* **27**, 1715-1721

8. Matsuyama, K., and Natori, S. (1988a) *J. Biol. Chem.* **263**, 17112-17116

9. Dimarcq, J. L., Zachary, D., Hoffmann, J. A., Hoffmann, D., and Reichhart, J. M. (1990) *EMBO J.* **9**, 2507-2515

10. Bakula, M. (1970) *J. Insect Physiol.* **16**, 185-197

11. Bakula, M. (1971) *J. Insect Physiol.* **17**, 313-319

12. Boman, H. G., Nilsson, I., and Rasmuson, B. (1972) *Nature* **237**, 232-234

13. Robertson, M., and Postlethwait, J. H. (1986) *Dev. Comp. Immunol.* **10**, 167-179

14. Flyg, C., Dalhammar, G., Rasmuson, B., and Boman, H. G. (1987) *Insect Biochem.* **17**, 153-160

15. Kylsten, P., Samakovlis, C., and Hultmark, D. (1990) *EMBO J.* **9**, 217-224

16. Keppi, E., Zachary, D., Robertson, M., Hoffmann, D., and Hoffmann, J. A. (1986) *Insect Biochem.* **16**, 395-402

17. Mason, P. J., and Williams, J. G. (1985) in *Nucleic Acid Hybridization: A Practical Approach* (Hames, B. D., and Higgins, S. J., eds) pp. 113-138, IRL Press, Oxford

18. Sanger, F., and Coulson, A. R. (1975) *J. Mol. Biol.* **94**, 441

19. Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) *Gene* (Amst.) **33**, 103-119

20. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

21. Auffray, C., and Rougeon, F. (1980) *Eur. J. Biochem.* **107**, 303-314

22. Corces, V., Pellicer, A., Axel, R., and Meselson, M. (1981) *Proc. Natl. Acad. Sci. U. S. A.* **78**, 7038-7042

23. Thomas, P. S. (1980) *Proc. Natl. Acad. Sci. U. S. A.* **77**, 5201-5205

24. Gilman, M. (1987) in *Current Protocols in Molecular Biology* (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds) pp. 4.7.1-4.7.8, John Wiley & Sons, New York

25. Cherbas, L., Schulz, R. A., Koehler, M. M. D., Savakis, C., and Cherbas, P. (1986) *J. Mol. Biol.* **189**, 617-631

26. Reichhart, J. M., Essrich, M., Dimarcq, J. L., Hoffmann, D.,

<footnote n="2">²S. C. Sun, I. Lindström, J. Y. Lee, and I. Faye, *Eur. J. Biochem.*, submitted for publication.</footnote>
Hoffmann, J. A., and Lagueux, M. (1989) *Eur. J. Biochem.* **182**, 423-427

27. Kockum, K., Faye, I., van Hofsten, P., Lee, J. Y., Xanthopoulos, K. G., and Boman, H. G. (1984) *EMBO J.* **3**, 2071-2075

28. Gunne, H., Hellers, M. and Steiner, H. (1990) *Eur. J. Biochem.* **187**, 699-703

29. von Heijne, G. (1986) *Nucleic Acids Res* **14**, 4683-4690

30. Boman, H. G., Boman, I. A., Andreu, D., Li, Z.-Q., Merrifield, R. B., Schlenstedt, G., and Zimmermann, R. (1989) *J. Biol. Chem.* **264**, 5852-5860

31. Kreil, G. (1990) *Trends Biochem. Sci.* **15**, 23-26

32. van Hofsten, P., Faye, I., Kockum, K., Lee, J. Y., Xanthopoulos, K. G., and Boman, I. A. (1985) *Proc. Natl. Acad. Sci. U. S. A.* **82**, 2240-2243

33. Matsumoto, N., Okada, M., Takahashi, H., Qu, X. M., Nakajima, Y., Nakanishi, Y., Komano, H., and Natori, S. (1986) *Biochem. J.* **239**, 717-722

34. Suchaneck, G., and Kreil, G. (1977) *Proc. Natl. Acad. Sci. U. S. A.* **74**, 975-978

35. Nakajima, Y., Xian-ming, Q., and Natori, S. (1987) *J. Biol. Chem.* **262**, 1665-1669

36. Li, Z., Merrifield, R. B., Boman, A., and Boman, H. G. (1988) *FEBS Lett.* **231**, 299-302

37. Samakovlis, C., Kimbrell, D. A., Kylsten, P., Engström, A., and Hultmark, D. (1990) *EMBO J.* **9**, 2969-2976

38. Matsuyama, K., and Natori, S. (1988) *J. Biol. Chem.* **263**, 17117-17121