Expression and characterization of cDNAs for Cecropin B, and antibacterial protein of the silkworm, Bombyx mori

Insect Btochem Molec Btol Vol 23, No 2, pp 285-290, 1993 Printed m Great Britain All rights reserved

0965-1748/93 $6 00 + 0 00 Copyright © 1993 Pergamon Press Ltd

Expression and Characterization of cDNAs for Cecropin B, an Antibacterial Protein of the Silkworm, Bombyx mori YUSUKE KATO,* KIYOKO TANIAI,* HIROHIKO HIROCHIKA,t MINORU YAMAKAWA*':~ Recewed 29 May 1992, revised and accepted 8 September 1992

To analyze the induction mechanism of antibacterial protein gene expression, cDNAs coding for cecropin B have been cloned from the B. mori fat body eDNA library. Nucleotide sequences of two positive clones were determined and their amino acid sequences deduced. They revealed that these clones coded for the same cecropin, which is identical to purified cecropin B. However, the cDNAs contained different nucleotides at the third codon position and 5' or 3' non-coding regions. Results obtained by Northern blot analysis showed that the gene expression of B. mori cecropin B was rapidly induced by Escherichia coli and reached maximum levels 8 h after immunization. The expression of cecropin B gene occurred specifically in tissues, mainly in the fat body and hemocytes. Antibacterial protein Molecularcloning cDNA lmmumty

Sequencing Tissue specific gene expression Insect

To solve such problems, we used the silkworm,

INTRODUCTION

B. mori, mainly because it has been a model ammal

The insect self-defense system contains an acute-phase induction of different proteins including lectin (Komano et al., 1980; Takahashi et al., 1985; Nanbu et al., 1991; Sugiyama and Natori 1991) and antibacterial proteins (Hoffman et al., 1981; Steiner et al., 1981; Hultmark et al., 1982; Qu et al., 1982; Dickinson et al., 1988; Okada and Natori, 1985; Kylsten et al., 1990). Cecropins, one group of antibacterial proteins, are known to be strong basic proteins with a molecular weight around 4000. They are also known to have a very broad spectrum against Gram-positive and -negative bacteria (Boman, 1986; Boman and Hultmark, 1987). Various msect species have been reported to induce cecropin synthesis by bacteria (Hoffman et al., 1981; Flyg et al., 1987; Robertson and Postlethwait, 1986). Furthermore, components of the bacterial cell wall, such as lipopolysaccharide and peptidoglycan, were identified to be possible triggers (De Verno et al., 1984; Samakovlis et al., 1990; Dunn et al., 1985). However, details on the generating mechanism of such triggers in connection with bacterial infection are not yet clarified. Moreover, the signal transduction mechanism in cells of a specific tissue, m which the cecropin gene expression occurs, also remains unclear.

for studies on gene regulations (Goldsmith and Kafatos, 1984; Suzuki and Suzuki, 1988), and partly because pathogenic information on this insect has been well established due to its importance in sericultural industries. We first tried to isolate cDNA clones for cecropins from a B. mori fat body cDNA library to understand the induction mechanism of the gene expression of antibacterial proteins. Here, we report characteristics of two cDNA clones encoding for B. mori (Bm) cecropin B and the analysis of the site and induction kinetics of the gene expression.

MATERIALS AND METHODS Biological materials

Silkworms, B o m b y x mori (Tokai x Asahi) were reared on an artificial diet (Nihonnosanko) at 25°C. Escherwhta coli (E. coli) K12 strain DH1 (Low, 1968; Meselson and Yuan, 1968), grown in LB medium (Maniatis et al., 1982) at 37°C with shaking, was used in injections into silkworms. Construction o f c D N A library

Fat bodies of fifth instar larvae, mjected with E. coli were isolated 9 h after immunization *Laboratory of BiologicalDefense,National Institute of Sericultural with the bacteria. Poly(A ÷ ) RNA was prepared from the and EntomologicalSoence, Tsukuba, Ibarakl 305, Japan tDepartmentof MolecularBiology,NationalInstituteof Agrobiologlcal fresh fat bo&es using a "Quick prep mRNA purification kit" (Pharmacia LKB). 8/~g of poly(A ÷) RNA were Resources, Tsukuba, Ibaraki 305, Japan :~Author for correspondence used for cDNA synthesis with a "cDNA synthesis kit" (4 × 10 6 cells/larva),

285

286

YUSUKE KATO et al

(Pharmacta LKB). cDNA was ligated with 2gtl0 vector (Murray et al., 1977). In vttro packaging was performed using a "GigapacklI gold packagmg extract" (Stratagene). Escherichia coli NM514 cells were transfected with recombinant viruses, giving a titer of 3.2 × 1 0 6 pfu/#g cDNA.

Nucleotide sequences of cecropin B cDNAs were compared using the secondary &mensional dot matrix by the SDS~Genetyx Harr plot analysis program. The stringency was set so that one dot denoted a match if 10 out of 15 consecutive nucleotides were identical.

Probe preparation

Northern blot analysis

A probe was prepared by polymerase chain reaction (PCR) (Salki et al., 1985; Mullis and Faloona, 1987) with 40 ng cDNA, Taq D N A polymerase (Promega) and synthesized primers. Primers were prepared based on the amino a o d sequences of Bm cecropin B (Teshima et al., 1986, 1987; Morishima et al., 1990). For 5' primer (18 mer) nucleotide sequences were deduced from Trp(2) to Lys(7) of the mature protein, and for 3' primer (14mer) from Pro(24) to Val(28). The sequences were (5')TGGAA(A/G)AT(A/C/T)TT(C/T)AA (A/G)AA(A/G) (Y) and ( 5 ' ) A C ( T / C ) T C ( A / G / T ) A T ( A / G / T / C ) G C (A/ G/T/C) GG(Y) for 5' and 3' primer, respectively. Thirty cycles of PCR were performed at 40°C for the annealing temperature by program temperature control system PC-700 (Astec). Nucleotlde sequences of the DNA fragment made by PCR were determined by the dideoxynucleotlde cham termination method (Sanger et al., 1977).

Fifth lnstar silkworm larvae in their third or fourth day were kept on ice m order to paralyze them, and injected in the abdominal leg wtth 20 #1 of E. coli strain DH1 (1 × 108 cells/ml of insect physiological sahne containing 150 mM NaC1 and 5 mM KC1). Fat bodies, sdk glands, hemocytes, midguts and Malpighian tubes were excised 20h later. A total RNA was extracted by the guanidium thiocyanate-cecium chloride method (Chirgwin et al., 1979). RNA was electrophoresed in 1.2% agarose containing 6.6% formaldehyde (Maniatis et al., 1982) and transferred onto "Gene Screen Plus" membranes (DuPont-New England Nuclear). A Bm cecropin B clone (BmcecB19) was used as a probe. A genomic D N A clone of Bm 18S rRNA, designated as pBmR161 (Maekawa et al., 1988) was also used as a

Plaque hybrMtzatton

Dot matrix analysis

1

2

3

Plaques (1 x 105) were transferred onto membrane filters (Schlelcher and Schuell) and screened with Bm cecropln B cDNA fragment prepared by PCR. The D N A fragment was labeled with [ct-3Zp]dCTP (ICN) and a " D N A labeling kit" (Nippon Gene). The prehybridization was performed for 4 h at 42°C in a solution containing 5 × Denhardt's solution, 5 × SSPE (Maniatis et al., 1982), 0.1 mg/ml salmon sperm DNA and 50% formamlde. Membrane filters were then hybridized with the probe for 16 h at the same temperature. The filters were washed twice with 2 x SSC (Maniatis et al., 1982) at room temperature for 5 min and twice w~th 0.2 × SSC containing 0.1% SDS at 60°C for 30 min before exposure to X-ray film. Single positive plaques were confirmed by repeating plaque hybridization three times under the same conditions as described above. Nucleotlde sequencing

cDNA inserts were separated by 2% low temperature melting agarose gel electrophoresis after &gestion with EcoRI and ligated to M13mpl8 phage cloning vector (Yanisch-Perron et al., 1985) cut with EcoRI. The hgated vectors were used to transform E. coli strain XL 1-blue. Single-stranded D N A was purified from recombinant M I3 phage and sequenced by the chain termination method (Sanger et al., 1977) using the universal M13 sequencmg primer and synthetic primers. The DNA fragments were labeled with [~-32p]dCTP (ICN) and Sequenase in the presence of A-, C-, G- or T-specific dideoxynucleotide mixtures (Umted States Biochemical Corp.). The radlolabeled fragments were electrophoresed in the 6% sequencing polyacrylamtde gel.

FIGURE 1 Gel electrophoresls of the synthesized DNA by polymerasechain reaction method Details of the synthesisconditions are described in the Materials and Methods section DNA was analyzed by 4% agarose gel electrophoresis Lane 1: Molecular size marker (~b X174 phage DNA &gested with HmclI) DNA fragment sizes are 1057, 770, 612, 495, 392, 345, 341, 335, 297, 291, 210. 162 and 79 bp Lanes 2 and 3 DNA synthesized by PCR. 1 #g of DNA was loaded onto each lane An arrow m&cates the position of 80 bp

C E C R O P I N B cDNAs F R O M B M O R I

probe for internal control. Autoradiographs were quantified by laser densitometer (LKB2222, LKB) scanning autoradiographs. For kmetic experiments, fat bodies were excised at the indicated time intervals after injection with E. coli as described above. RNA extraction and other conditions were the same as already mentioned. RESULTS

AND DISCUSSION

Knowing the amino acid sequences of Bm cecropins (Tesh]ma et al., 1986, 1987; Morishima et al., 1990), we took advantage of the data to prepare a probe for screening the cDNA library. We first synthesized d~fferent primers for PCR and found that a combinat]on of primers, which encode for amino acid sequences of Bm cecropin B mature protein from Trp(2) to Lys(7) and from Pro(24) to Val(28), gave a clear DNA band on an agarose gel electrophores~s, with the expected chain length (Fig. 1). Nucleotide sequences of this D N A fragment confirmed that they encode for the mature protein region of Bm cecropm B from Trp(2) to Val(28) (data not shown). We then used the D N A fragment to screen a full length cDNA for Bm cecropin B. One thousand positive clones were obtained from 1 × 105 plaques, indicating that cecropin-related m R N A occupies 1% of the total mRNA. Size analysis of cDNA inserts from positwe clones showed that about 400 base pair (bp) cDNAs were the mam isolates. We chose two of the clones designated

287

as BmcecB19 and -21 and determined their nucleotide sequences. As the size of cDNAs was short, we first determined the sequences using universal primers for the M13mpl8 vector. Then, primers based on the determined sequences were synthesized on an ABI 381 A D N A synthesizer. These primers were used to further determine the rest of the sequences. The complete nucleotide and deduced amino acid sequences of BmcecB19 and -21 clones are shown in Fig. 2. The sequences contained an open reading frame and consisted of 420 and 412 nucleotides for BmcecB19 and -21, respectively. The first A T G codons were seen at 54 bp (BmcecB19) and 50 bp (BmcecB21) downstream from the EcoRI linker site. The A T G start codon is followed by signal peptide sequences of 26 amino acid residues, a mature portion of Bm cecropin B and 2 additional amino acid residues which are removed during processing (Monshlma et aL, 1990). Processing of the carboxyl-terminal glycine residue to an amlde group is also known to occur m both Hyalophora cecropia (Hc) and Sarcophaga peregrina (Sp) (Kylsten et al., 1990). The translation termination codon (TGA) is located at positions 243-246 (BmcecB19) or 240-243 (Bmcec21). A putatwe recognition sequence (AATAAA) for add]tion of the poly(A ÷) tail (Proundfoot and Brownlee, 1976) was not observed in these clones. A poss]ble explanaUon for this lS that oligo dT, used as a 3' primer for the construction of the cDNA library, hybridized to the A rich reg]on of mRNAs instead of to the poly A site. An open reading frame encodes for

BmcecB19

~

BmcecB19 ~ R~rI'I~AA'rI-~aCAT~ACTTA(I~ATCa%ATTIE Bmeec~_~1 -GC . . . . . . . . . . . . . . . . . . . . . . . -GG- . . . . . . . . . . . . . . . . . . . . . . . . - C - - MetAsnPhe

BmcecB19 GCAAAC~TOCTA Ti'it~GCA Ba~gc~-rb2_l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

~

62 59 3

122 119

AI ~ r,ysI l e L e u S e r P h e V a l P h e A I~ T ~ u V a l I ~ M e t T n r S e r A / a A l a

lMmecB19 ~ ~ BmcecB21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

C

PrcGluProArgTrpLysI lePheLysLysI leGluLysMetGlyArgA~al leArgAsp

23

182 179 43

A BmcecB19 GGGA AAAGCTATCGGAAAA Bmcec__B21 - - C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . GlyIleValLysAlaGly~aIleGluVa~lySerA]a T.ysAlaIleGlyLys

242 239 63

~I-J~ATI-I-I~.~T 302 ]imec_.B19 TGATC:r r J~AA~/3C~.CIG C C A ~ ~ A ~ T I ~ A A T L - ~ . A C I t Bmcec_ _R:,2_1 . . . . A . . . . . . . . . T A - A . . . . . . . . - C . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . 299

BmcecB19 ~~ACAGcrI"I"A/~C4~FI~TAATAAGACATIEAA'rrI"n~AATATAATI~ l]mo~__R2_l . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . -G- . . . . . . . . . . . . . . . . . . .

362 359

BmoecB19 Bmc~7_ _R21

420

-1-ri-it. ~ 3 G I V ~ T A A A O d r J ~ T G T I G C A A A A T F A A A A A . . . . . . . . . . . . . . . . . . . . . . -C . . . . . . . . . . -G- . . . . . . . . . T . . . . . .

412

F I G U R E 2 Nucleotlde and the deduced amino acid sequences of BmcecBl9 and BmcecB21. The termination codon is marked with asterisks An arrow mdlcates the N-terminal of the mature protein Dashed hnes indicate nucleoUdes ~dent]cal with those of BmcecBl9

288

Y U S U K E K A T O et al

63 codons and the deduced amino acid sequences are identical to those of Bm cecropin B determined from the direct analysis of the purified protein (Teshima et al. 1986, 1987; Morishima et al. 1990). However, nucleotide sequences of BmcecB 19 and -21 contain different nucleotides at the third position of the codons and 5' or 3' non-coding regions. The deduced amino acid sequences showed that there is no N-glycosylation site (Asn-X-Ser or Asn-X-Thr) in these clones, indicating a typical characteristic of cecropins. Homology of the nucleotide sequences of Bm cecropin B was compared with those from other insects. Figure 3 shows the dot matrix analysis between Bm cecropin B (BmcecB19) and either Hc cecropin B (Van Hofsten et al., 1985) or Drosophila melanogaster (Dm) cecropin B (Kylsten et al., 1990). Results clearly reveal that the nucleotide sequence of Bm cecropin B is highly conserved in Hc cecropin B, and to a lesser extent, in Dm cecropin B. Scores of identity in the nucleotide sequence between cecropin B of B. mori and H. cecropia or D. melanogaster were estimated to be 71.3 and 47.2%, respectively• According to conventional classificatton, (A) 1

101

:>01

301

1

,,,', ~. -. "~.~

••

iOl

401

"

",

~,'

%1

•

201

\

..

• ,~..

B. mori and H. cecropia belong to the same order, Lepidoptera, while D. melanogaster belongs to the order Diptera. Thus, the homology in the sequences of lepidopteran cecropin B indicates a very close relationship in the evolutionary system. A number of tissues from B. mori were exammed for their capaoty to synthesize cecropin B. The total RNA was isolated from Malpighian tubes, midguts, hemocytes, silk glands and fat bodies. Figure 4 shows the results of Northern blot analysis. The results indicate that fat body and hemocytes are the main sites where cecropin B gene expression occurs. It is noteworthy that the intensity of cecropin B gene expression in hemocytes was almost the same as in the fat body. In the case of M. sexta (Dickinson et al., 1988), Northern blot analysis showed that cecropin gene was expressed mainly in the fat body, but all other tissues, including hemocytes, showed positive signals, although the intensity was extremely weak compared with that from the fat body. These two lepidopteran species, B. mort and M. sexta, therefore, showed a significant difference in gene expression in hemocytes. In a dipteran insect, D. melanogaster, in situ hybridization analysis showed that cecropin A and B genes were expressed strongly in the fat body and hemocytes (Samakovlis et al., 1990). From the aspect of the dominancy of hemocytes in the expression of cecropin gene, the tissue specificity in B. mori resembles that in D. melanogaster. Cecropin B gene expression was also observed in other tissues such as silk glands, midguts and Malphigian tubes, but to a far lesser extent. This result was similar to the case of M. sexta, however, we could not exclude the possibility of slight contamination by hemocytes under our experimental conditions.

• .~,

150 122

(B) 101

1

201

30!

401

1

re

%

"%

101

~

"

v

•

?

100

100

•

t~ O

..~',

_>

50

re 201

"N

%

•

301

•t: •x

•'

:"

, ' • ", ,

•

"

. 1:

401 F I G U R E 3 D o t matrix analysis o f nueleoUde sequences o f cecropin B eDNAs. D o t matrix analysis between Bm cecropm B and either Hc ceeropm B or D m cecropin B was carried out as described in the Materials and Methods section The horizontal axes represent the sequence from Bm eecropin B (BmcecB19 clone) and the vertical axes are the sequences of eeeropln B c D N A s from H cecropta (A) and D melanogaster (B)

0.65

1.75

2.40

SG

MG

MT

HC

FB

F I G U R E 4 Tissue distribution of cecropin B gene transcripts Total R N A isolated from various tissues was electrophoresed and transferred to nylon membranes Membranes were hybn&zed with a riP-labeled Bm cecropln B e D N A (BmcecB19) probe. After autoradiography, secondary hybridization was carried out with a Bm 18S r R N A probe (pBmR161) and the membrane was exposed again to X-ray film. To quantify the intensity of hybridized signals, autora&ographs were scanned with a densltometer Intensity values of cecropm B m R N A (Rcec) were normahzed against those of 18S r R N A (R18S) and presented as relatwe values, where the value of fat body is 100 Numbers on the top of each column indicate relative values SG, silk gland, M G , mldgut, MT, Malpighian tube, HC, hemocytes, FB, fat body

CECROPIN B cDNAs FROM B M O R I

1

2

3

4

5

289

6

7

FIGURE 5. Time-course of Bm cecropm B gene expression after inducUon. Fat bodies were excised at 0 (lane I), 2 (lane 2), 4 (lane 3), 8 (lane 4), 12 (lane 5), 16 (lane 6) and 24 h (lane 7) after Immunization with E. coh. 10/lg of total RNA were electrophoresed and Northern blot hybridization was performed with a Bmcecl9 cDNA clone.

A time-course of accumulation of cecropin B gene transcripts was investigated after immunization. For this, fat body was excised at various time intervals after injection of live E. coli and total RNA sample was analyzed by Northern blot hybridization. As is seen in Fig. 5, the transcripts were detectable by 2 h, reached a maximum abundance at 8 h and gradually decreased thereafter. In the case of M. sexta, S. peregrina and D. meranogaster, the time to reach a maximum level of accumulation was 8-12, 6 and 6h, respectively (Dickinson et al., 1988; Matsumoto et al., 1986; Kylsten et al., 1990). These results reveal that the induction occurs rapidly after immunization and is due to transcriptional regulation, or post-transcriptional control such as mRNA stabilization and not to translational event. In summary, this work has characterized two cDNA clones encoding Bm cecropin B. Cecropin B gene expresses tissue specifically mainly in the fat body and hemocytes. The gene expression is induced in a short time by live E. coli, indicating an efficient self-defense response against bacteria. REFERENCES Boman H. G (1986) Antzbacterml immune proteins in insects Syrup zooL Soc Lond 56, 45-58. Boman H. G. and Hultmark D. (1987) Cell-free immumty m insects A Rev. Mwroblol 41, 103-126. Chirgwin J. M., Przybyla A. E., MacDonald R J and Rutter W J (1979) Isolation of biologically active nbonucletc acid from sources enriched m rlbonuclease. Biochemistry 18, 5294-5299. De Verno P. J., Chadwick J. S., Aston W P and Dunphy G B (1984) The m vitro generatzon of an anUbacterml actlwty from the fat body and hemolymph of non-zmmumzed larvae of Gallerta mellonella Dev. comp Immunol 8, 537-546 Dlckson L , Russel V and Dunn P. E (1988) A family of bacteriaregulated, cecropm D-hke peptzdes from Munduca sexta J biol. Chem 263, 19424-19429 Dunn P E , Dal W., Kanost M. R and Geng C. (1985) Soluble pepUdoglycan fragments stzmulate antibacterial protein syntheszs IB 23/2--D

by fat body from larvae of Manduca sexta Dev comp. lmmunol. 9, 559-568. Flyg C., Dalhammar G., Rasmuson B. and Boman H G. (1987) Insect Immunity. Inducible antibacterial activity in Drosophda Insect Btochem. 17, 153-160. Goldsmith M. R. and Kafatos F C (1984) Developmentary regulated genes m Silkworm. A. Rev Genet 18, 443-487. Hoffman D , Hultmark D and Boman H. G. (1981) Insect lmmumty: Gallerta mellonella and other lepidoptera have cecropla-P9-1ike factors active against Gram-negative bactena. Insect Btochem 11, 537-548 Hultmark D , Engstrom ,~., Benmch H , Kapur R. and Boman H G (1982) Insect immunity" isolaUon and structure of cecropin D and four minor antibacterial components from cecropla pupae Eur. J Btochem. 127, 207-217 Kaaya G P , Flyg C. and Boman H G (1987) Insect immunity. Induction of cecropin and attacm-hke antibacterial factors m the haemolymph of Glossma morsitans morsttans Insect Blochem 17, 309-315 Komano H , Mizuno D and Natori S. (1980) Punfication of lectm induced In the hemolymph of Sarcophaga peregrlna larvae on injury J biol Chem 255, 2919-2924. Kylsten P , Samakovhs C and Hultmark D (1990) The cecropin locus in Drosophda, a compact gene cluster involved m the response to infection EMBO J 9, 217-224 Low B (1968) Formation of merodzploids in matlngs with a class of rec- recipient stratus of Eschertchta cob K12. Proc. natn Acad. Sct U S.A 60, 160-167 Maekawa H., Takada N , Mlkltam K., Ogura T , Mtyajlma N., Fujiwara H , Kobayashl M. and Nlnakl O (1988) Nucleolus organtzers m the wdd sdkworm Bombyx mandarma and the domesticated sdkworm B. mort Chromosoma (Berl.) 96, 263-269 Mamatls T , Fntsch E F and Sambrook J (1982) Molecular Cloning. A Laboratory Manual Cold Spnng Harbor, New York. Matsumoto N , Okada M , Takahashl H., Mlng Q. X , Nakajlma Y , Nakamshl H , Komano H and Naton S (1986) Molecular cloning of a cDNA and assignment of the C-terminal sarcotoxm IA, a potent anUbacterml protein of Sarcophaga peregrma Btochem J 239, 717-712 Meselson M and Yuan R. (1968) DNA restriction enzyme from E coh. Nature 217, Ill0-1114 Monshlma I , Suglnaka S, Ueno T and Hirano H. (1990) Isolation and structure of cecroplns, inducible antibacterial peptides, from the silkworm, Bombyx mort. Comp. Blochem Physiol 95B, 551-554 Mulhs K B and Faloona F A. (1987) Specific synthesis of DNA

290

YUSUKE KATO et al

in vitro wa polymerase catalyzed chain reaction Methods Enzymol 155, 335 350 Murray S. E , Brammar W J and Murray K (1977) Lambdold phages that simplify the recovery of m vitro recombmants Molec gen. Genet 150, 53-61 Nanbu R , Kubo T , Ueno K and Naton S (1991) Preferentml deadenylation of Sarcophaga lectm mRNA during its acute phase expression Btochtm btophys Acta 1088, 71 76 Okada M and Naton S (1985) Primary structure of sarcotoxm I, an antibacterial protein reduced in hemolymph of Sarcophaga peregrma (flesh fly) larvae J btol Chem 260, 7174-7177 Proudfoot N J and Brownlee G G (1976) 3' Non-coding region sequence m eukaryotic messenger RNA Nature 263, 211-214 Qu X - m , Sterner H , Engstrom /~, Bennlch H and Boman H G (1982) Insect lmmumty isolation and structure of cecropm B and D from pupae of the Chinese oak silk moth, Antherea pernyt Eur J Bzochem 127, 219-224 Robertson M and Postlethwait J H (1986) The humoral antibacterml response of Drosophtlla adults Dev comp lmmunol 10, 167 179 Salkl R K , Scharf S., Faloona F,, Mulhs K B, Horn G T, Erhch H A and Arnheim N (1985) Enzymatic amplification of fl-globm genomic sequences and restriction site analysis for dmgnosls of sickle cell anemia Science 230, 1350 1354 Samakovhs C, Klmbrell D A , Kylsten P, Engstrom/k and Hultmark D (1990) The immune response m Drosophila pattern of cecropm expression and biological actwlty EMBO J 9, 2969-2976 Sanger F , Nlcklen S and Coulson A R (1977) DNA sequencing with chain terminating mhibltors Proc natn Aead Sct U S A 74, 5463 5467 Spies A G , Karhnsey J E and Spence K D (1986) Antibacterial hemolymph proteins of Manduca sexta Comp Btoehem Phystol 83B, 125-133 Sterner H , Hultmark D , Engstrom/~, Benmch H and Boman H G (198 I) Sequence and specificity of two antibacterial proteins involved

m insect lmmumty Nature 292, 246-248 Suglyama H and Naton S (1991) A novel mechamsm of Satcophaga lectln gene expression Possible involvement of oxidation of the sulfhydryl group of a fat-body protein Eur J Btochem 200, 495 500 Suzuki T and Suzuki Y (1988) Interaction of composite protein complex with the fibrom enhancer sequence J btol Chem 263, 5979 5986 Takahashi H , Komano H , Kawaguchi N , Kitamura N , Nakamshi S and Naton S (1985) Clonmg and sequencing of cDNA of Sarcophaga peregrma humoral lectm induced on inJury of the body wall J biol. Chem 260, 12228-12233 Teshima T., Nakal T , Uekl Y and Shlba T (1987) Structure determination of lepidopteran C, self-defence substance produced by sdkworm (Bombyx mort) Tetrahedron 43, 4513-4518 Teshima T , Uekl Y , Nakai T , Shlba T and Klkuchi M (1986) Structure deterrmnatlon of lepidopteran, self-defense factor produced by silkworm Tetrahedron 42, 829-834 Van Hofsten P , Faye I , Kockum K , Lee J -Y, Xanthopoulos K G , Boman I A , Boman H G , Engstrom/k, Andreu D and Mernfield R B (1985) Molecular cloning, cDNA sequencing, and chemical synthesis ofcecropm B from Hyalophora ce~ropm Proc nam Aead Sci U S .4 82, 2240-2243 Yamsch-Perron C , Vleira J and Messing J, (1985) Improved MI3 phage vectors and host strains nucleotide sequence of the M l3mpl8 and pUCI9 vectors Gene 33, 103-119

Acknowledgements--We are indebted to Dr Htdeakl Maekawa, National Institute of Health, Japan for the gift ofpBmRl61 This work was supported in part by a Grant-m-Aid (Blo Media Program) of the Ministry of Agriculture, Forestry and Fisheries, Japan (BMP 93-IV2-1) and by a special coordination fund for promoting science and technology (SCF) m the basic research core system by the Science and Technology Agency (STA), Japan