a bombyx mori gene, bmchi-h, encodes a protein homologous to bacterial and baculovirus chitinases

Insect Biochemistry and Molecular Biology 33 (2003) 749–759www.elsevier.com/locate/ibmb

A Bombyx morigene,BmChi-h, encodes a protein homologous tobacterial and baculovirus chitinases

Takaaki Daimona, Koutaro Hamadaa,1, Kazuei Mitab, Kazuhiro Okanoc,2,Masataka G. Suzukia,c, Masahiko Kobayashia, Toru Shimadaa,∗

a Laboratory of Insect Genetics and Bioscience, Department of Agricultural and Environmental Biology, Graduate School of Agricultural andLife Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan

b Genome Research Department, National Institute of Agrobiological Sciences, Owashi 1-2, Tsukuba, Ibaraki 305-8643, Japanc Laboratory of Molecular Entomology and Baculovirology, Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako,

Saitama 351-0198, Japan

Received 19 March 2003; accepted 20 May 2003

Abstract

We have cloned and characterized a novel chitinase gene (BmChi-h) from the silkworm,Bombyx mori. BmChi-hcDNA has anopen reading frame of 1665 nucleotides, encoding a protein of 555 amino acid residues. The predicted protein shared extensivesimilarities with bacterial and baculovirus chitinases in both amino acid sequences (73% identity withSerratia marcescenschiAand 63% withAutographa californicanucleopolyhedrovirus chiA) and domain architectures.BmChi-hwas a single-copy gene andlocated on chromosome 7. The expression ofBmChi-hmRNA was observed in a stage- and tissue-specific manner that was almostidentical to that of another chitinase gene previously cloned fromB. mori. We further determined the overall genomic organizationof BmChi-h. There was no intron in the ORF ofBmChi-h. However,BmChi-hwas transcribed from three promoters, which generatedthree isoforms in the 5�-UTR of the transcript. Phylogenetic analysis suggested that ancestral species ofB. mori acquired thechitinase gene from a bacterium or an ancestral baculovirus via horizontal gene transfer. 2003 Elsevier Ltd. All rights reserved.

Keywords:Chitinase; Horizontal gene transfer;Bombyx mori; Expressed sequence tag (EST); Baculovirus;Serratia marcescens

1. Introduction

Chitin, a 1,4-β-linked polymer of N-acetyl-β-d-glu-cosamine, is the second largest bio-polymer next tocellulose. Chitin has been found in the integuments ofarthropods, nematodes, and molluscs, the gut linings ofinsects, the cell walls of fungi and some algae, and theshells of crustaceans. Chitinolytic enzymes that catalyzethe hydrolysis of chitin have been found not only in chi-tin-containing organisms but also in bacteria, plants, ani-mals, and viruses that do not contain chitins. Chitinases

∗ Corresponding author. Tel.:+81-3-5841-8130; fax:+81-3-5841-8011.

E-mail address:shimada@ss.ab.a.u-tokyo.ac.jp (T. Shimada).1 Present address: Asahi Breweries Co. Ltd., Midori 1-1-21,

Moriya, Ibaraki 302-0106, Japan.2 Present address: Department of Microbiology, Oregon State Uni-

versity, Nash Hall 220, Corvallis, OR 97331-3804, USA.

(EC 3.2.1.14) belong to families 18 and 19 of glycosylhydrolases, which are different in their amino acidsequences and their catalytic mechanisms (Henrissat,1991, 1999). Even within the same family, they exhibitgreat diversity and variations in their sequence, domainstructure, and enzymatic properties. Chitinases are pro-duced in many organisms for each biological function,such as molting of the exoskeleton in insects and crus-taceans, cell growth and division in fungi, degradationof chitin for nutrients in bacteria, and self-defence inplants. Some pathogens of chitin-containing organismsare believed to utilize chitinolytic enzymes for pen-etration into the host body or release of their progeny(Kramer and Muthukrishnan, 1997; Gooday, 1999).

In insects, chitinases are induced stage- and tissue-dependently to degrade the chitin in the exoskeleton andperitrophic membrane (Kramer et al., 1993; Kramer andMuthukrishnan, 1997), and chitinase genes have beencloned from various insects. In lepidopteran insects, chi-

750 T. Daimon et al. / Insect Biochemistry and Molecular Biology 33 (2003) 749–759

tinase cDNAs were cloned from Manduca sexta(Krameret al., 1993), Bombyx mori (Kim et al., 1998),Hyphantria cunea(Kim et al., 1998), Spodoptera litura(Shinoda et al., 2001), and Choristoneura fumiferana(Zheng et al., 2002). These lepidopteran chitinases exhi-bit extensive similarity in both their amino acidsequences (70–80% identities to each other) and domainstructures. The overall structures of chitinase genes havebeen determined in those from M. sexta (Choi et al.,1997) and B. mori (Mikitani et al., 2000; Abdel-Banatand Koga, 2001), and their genomic organization isalmost identical (Abdel-Banat and Koga, 2001). Thecopy number of the chitinase gene of B. mori has beenestimated as one (Mikitani et al., 2000; Abdel-Banat andKoga, 2001), while there are at least four chitinase genesin dipteran insects (de la Vega et al., 1998).

Large-scale sequencing of expressed sequence tags(ESTs) of B. mori has been performed in the Bombyxgenome project (SilkBase; http://www.ab.a.u-tokyo.ac.jp/silkbase/). We have roughly characterizedthem to identify novel chitinolytic enzymes and foundthat 46 ESTs showed homology to chitinases. Bysequencing and assembling these ESTs, we classifiedthem into five cDNAs that seemed to encode differentproteins. Among the five cDNAs, one was identical tothe chitinase gene previously cloned from B. mori (Kimet al., 1998), while the others seemed to be derived fromnovel chitinase or chitinase-like genes.

The most surprising finding was that the amino acidsequence of one novel chitinase showed extensive hom-ology to those of bacterial and baculovirus chitinases,especially S. marcescenschiA (73% identity) andAcNPV chiA (63% identity). It showed much lowerhomology to the chitinases from eukaryotes (E - value� 10�44 for fungal chitinases, �10�36 for arthropodchitinases) compared to those from bacteria and baculo-viruses (E - value = 0.0 for S. marcescenschiA andAcNPV chiA). The presence of such a chitinase gene inthe EST database of B. mori was quite interesting,because it has been proposed that an ancestral baculo-virus may have acquired the chitinase gene from a bac-terium via horizontal gene transfer (Hawtin et al., 1995).

In this study, we describe the cloning and characteriz-ation of a novel chitinase gene that shows extensivesimilarities to bacterial and baculovirus chitinases. Weperformed linkage analysis to prove that it actuallyexisted in the genome of B. mori and to determine itschromosomal localization. The expression profile of itstranscript was examined to demonstrate whether it isfunctional or not and predict its function. We furtherdetermined its overall genomic organization. The evol-utionary relationship between this novel chitinase andother chitinases was also discussed.

2. Materials and methods

2.1. Insects

We used Bombyx moristrains p50T, C108T, whichwere maintained in the University of Tokyo, Japan, andF1 hybrid Kinshu × Showa purchased from Ueda San-shu, Nagano, Japan. Specimens of the wild silkwormBombyx mandarina, which were collected locally andmaintained in the University of Tokyo, Japan, were alsoused. The larvae of Kinshu × Showa were reared on anartificial diet (Nosanko, Japan), and the others werereared on fresh mulberry leaves, in a conditioned insect-rearing room (25 °C, 12L:12D photoperiod). Tissuesexamined were dissected and washed in PBS (137 mMNaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mMKH2PO4) two times, immediately frozen in liquid nitro-gen, and stored at –80 °C.

2.2. Classification of ESTs showing homology tochitinase genes

ESTs showing homology to chitinases were searchedfor in the EST database of B. mori (SilkBase) using theBLAST program, and 46 ESTs were collected. Bysequencing and assembling the nucleotide sequences ofthese ESTs, we obtained five non-redundant cDNAs. Topredict the protein encoded by these cDNAs, homologysearch using BLASTX program against public databasewas performed. Among these five cDNAs, one cDNAthat showed high homology to bacterial and baculoviruschitinases was subjected to further analysis in this study.The nucleotide sequence was determined using the ABIBig Dye Terminator Cycle Sequencing Ready ReactionKit (Applied Biosystems, CA, USA) and an ABI Prism3100 DNA sequencer (Applied Biosystems, CA, USA).Sequence data were analyzed with the program packageGENETYX-WIN version 5.1 (Software DevelopmentCo., Tokyo, Japan).

2.3. 5�- and 3�-rapid amplification of cDNA ends(RACE)

Rapid amplification of cDNA ends (RACE) was car-ried out using a SMART RACE cDNA AmplificationKit (Clontech Laboratories, CA, USA). Poly(A) +RNAs extracted from wing disks of day-2 spinning lar-vae using the Micro Fast-Track 2.0 Kit (Invitrogen, CA,USA) were used as templates. Reverse transcription andadapter ligation were performed following the manufac-ture’s protocol. The 5�-end of the cDNA was PCR-amplified in an amplification reaction (50 µl) containing2.5 µl of 5�-RACE-Ready cDNA, 0.2 mM deoxyribonu-cleotides, 10 µM of a gene-specific primer GSPR1 (5�-CGGCCGCTGCATGAACGTTGTAGTGCTT-3�), 10µM of the Universal Primer Mix supplied in the kit, and

751T. Daimon et al. / Insect Biochemistry and Molecular Biology 33 (2003) 749–759

2 units of ExTaq DNA polymerase (TaKaRa, Japan).Temperature cycling was carried out at 94 °C for 3 min,followed by five cycles of 30 s at 94 °C and 3 min at72 °C, five cycles of 30 s at 94 °C, 30 s at 70 °C, and3 min at 72 °C, and 20 cycles of 30 s at 94 °C, 30 s at68 °C, and 3 min at 72 °C. The 3�-end of the cDNAwas PCR-amplified in an amplification reaction (50 µl)containing 2.5 µl of 3�-RACE-Ready cDNA, 0.2 mMdeoxyribonucleotides, 10 µM of a gene-specific primerGSPF1 (5�-GCAGGTCGAGCTCTGCAACTCAGACGG-3�), 10 µM of the Universal Primer Mix supplied in thekit, and 2 units of ExTaq DNA polymerase (TaKaRa,Japan). Temperature cycling was carried out under thesame conditions as for 5�-RACE. The PCR productswere cloned into a pGEM-T Easy Vector (Promega, WI,USA), and the nucleotide sequences were determined asdescribed above.

2.4. Phylogenetic analysis

Sequences homologies were analyzed using theBLAST program for public DNA/protein databases. Theamino acid sequences of the catalytic domains werealigned with the ClustalX program (Thompson et al.,1997). Phylogenetic trees were constructed withneighbor-joining methods using MEGA program version2.1 (Kumar et al., 2001)

2.5. Southern blot analysis

Genomic DNA was extracted from the posterior silkglands of mid-fifth instar larvae according to the protocoldescribed by Suzuki et al. (1998). Ten micrograms ofgenomic DNA of strain p50T and 2 µg of BAC DNAof clone 93-16L were fully digested with restrictionenzymes at 37 °C overnight and electrophoresed on a0.8% agarose gel. After depurination in 0.25 M HCl, thegel was neutralized in 0.4 M NaOH. Then, the gel wastransferred onto a nylon membrane (Hybond XL, Amer-sham, UK) in 0.4 M NaOH overnight, and the blottedmembrane was baked for 2 h at 80 °C. Prehybridization,hybridization, and washing were carried out as indicatedin the supplier’s protocol. Exposure, analysis, and print-ing were performed using a BAS2000 Bioimage Ana-lyzer (Fuji Photo Film, Japan). The DNA probe wasamplified by PCR with primers Chif3 (5�-GACTTCAAGGTTTCTATCCA-3�) and Chir3 (5�-AGGATTCCACACGTATGGAG-3�) using EST clonewdS20876 as a template and labeled with α-32P usingthe Random Primer DNA Labeling Kit (TaKaRa, Japan).

2.6. Linkage analysis

Linkage mapping was done using PCR–restrictionfragment length polymorphism (PCR–RFLP) between B.mori strain p50T and its related species, B. mandarina.

The genomic DNA of 30 individuals of (p50T × B.mandarina) × p50T was extracted, and the fragment ofBmChi-h and its homologue of B. mandarina was PCR-amplified with primer chif1 (5�-CGCGGATCGTCGAAGTCAACC-3�) and chir1 (5�-GCCATCACCACCACAAATCG-3�); the product was digested with DraI andelectrophoresed on a 2.0% agarose gel. Rcf47, a DNAmarker of chromosome 7 (Sugasaki, personalcommunication), was also PCR-amplified from the gen-omic DNA of the same 30 individuals, digested withMspI, and electrophoresed on a 2.0% agarose gel.

2.7. Northern blot analysis

Total RNA was extracted from F1 hybrid Kinshu ×Showa using an Easy Prep RNA Kit (TaKaRa, Japan),and 10 µg of each RNA were electrophoresed on 1.2%agarose gels containing 2.2 M formamide in a MOPSbuffer and transferred onto a Hybond XL nylon mem-brane (Amersham, UK). The filter was hybridized andwashed following the procedures described previously(Suzuki et al., 1998). The same probe as for Southernblot analysis was used. The same blot was rehybridizedsuccessively with probes for B. mori chitinase and B.mori Actin3. A probe for B. mori chitinase was amplifiedby PCR with primers ChiMF1 (5�-GGATGTCCTTCAT-CAGAAGCGT-3�) and ChiMR2 (5�-GGGTCTGATGGAGTTGAATTG-3�) using EST clone fbpv0443 as atemplate. A probe for B. mori Actin3 was amplified byPCR with primers BA3F1 (5�-AGATGACCCAGAT-CATGTTCG-3�) and BA3R1 (5�-GAGATCCA-CATCTGTTGGAAG-3�) using EST clone wdS20279 asa template.

2.8. Shotgun sequencing analysis of BAC clone

A silkworm high-density blot filter of the BAC library(Koike et al., 2003) was screened using the ECL DirectNucleic Acid Labeling and Detection System(Amersham, UK) with the same probe as used in South-ern blot analysis. BAC DNA of a positive clone, 93-16L,was isolated using the Large-Construct Kit (QIAGEN,Germany) and sheared to an average of 2 kb fragmentsusing Hydroshear (GeneMachines, CA, USA). Theresultant fragments were repaired with T4 DNA poly-merase and inserted into pUC18/HinC�/BAP. 1440clones were randomly picked up from the subcloninglibrary and sequenced with both M13-47 and RV-M pri-mers using the DYEnamic ET Dye Terminator Kit(Amersham, UK), and the nucleotide sequences weredetermined by MegaBACE1000 (Amersham, UK). Con-tigs were constructed from the raw sequence data usingCAP4 Sequence Assembly Software (Paracel, CA,USA).

2.9. Sequence deposition

The nucleotide sequence of BmChi-h was submitted tothe DDBJ/EMBL/GenBank databases with the accessionnumber AB104488. The nucleotide sequences of ESTsof B. mori are available at SilkBase (http://www.ab.a.u-tokyo.ac.jp/silkbase/) and DDBJ/EMBL/GenBank data-bases (AU000001–AU006458, AV398029–AV406327,and BP114820–BP128308).

3. Results

3.1. Classification of ESTs encoding chitinase orchitinase-like proteins

We obtained 46 ESTs that show homology to chitin-ase genes from the EST database of B. mori (SilkBase),and assembled them to five non-redundant cDNAs(Table 1). They were predicted to encode different pro-teins by homology search using the BLASTX program.Among five cDNAs, one was apparently identical to thechitinase gene previously cloned from B. mori (Table 1,EST-Chi1), while the others seemed to encode the novelproteins of B. mori (Table 1, EST-Chi2 to Chi5). Thesenovel proteins showed high homology to chitinases orchitinase-like proteins from other organisms. The mostsurprising finding was that one sequence (Table 1, EST-Chi2) exhibited extensive homology to chitinases frombacteria and baculoviruses (E - value = 0.0). Thus, thiscDNA was subjected to further analysis in this study.

3.2. Cloning of a novel chitinase cDNA of Bombyxmori

To obtain the full-length cDNA corresponding to theEST-Chi2 from B. mori, 5�- and 3�-RACE was carriedout with primer sets designed based on the nucleotidesequence of wdS20876, an EST clone of EST-Chi2(Table 1). When 5�-RACE was performed, three differ-ent types of nucleotide sequences were obtained (Fig.1A). They were different only in putative 5�-untranslatedregions but completely identical to one another in theircoding regions. The ESTs corresponding to each isoformwere found in SilkBase, suggesting that these isoformswere not artifacts but genuine transcripts of B. mori.Nucleotide sequences of more than 10 clones per iso-form were determined, and the most abundant sites ofthe 5�-ends were defined as the transcription start sitesof each isoform. When 3�-RACE was performed, a sin-gle type of the nucleotide sequence was obtained, inwhich a putative polyadenylation signal and poly-Asequence were found (Fig. 1B). Although there are threevariants in the 5�-UTR of the cDNA, the nucleotidesequence of their protein-coding regions was identical.

3.3. Characterization of the cDNA and comparisonswith other chitinases

The cDNA had an open reading frame of 1665 nucleo-tides, encoding a protein of 555 amino acid residues(Fig. 1). The N-terminal sequence of this predicted poly-peptide consisted of a signal peptide containing hydro-phobic residues. Signal P prediction positioned the puta-tive cleavage site between residues 20 and 21. Proteinpattern search against the Prosite database indicated thatthe protein belongs to family 18 chitinases, which com-prise microbial, arthropod, nematode, mammalian, andsome plant chitinases. The active site signature of thefamily 18 chitinases (Van Scheltinga et al., 1994) ishighly conserved and located at the amino acid residues302–310, FDGVDIDWE.

There were extensive similarities between the proteinand the chitinases of bacteria and baculoviruses in theiramino acid sequences and domain structures (Fig. 2A).The amino acid sequence of the protein showed 73%identity to S. marcescens chiA (Jones et al., 1986) and63% to AcNPV chiA (Hawtin et al., 1995). In contrast,the protein showed much lower sequence similarities toknown chitinases of insects (27% identical to B. mori[U86876] and 26% to Manduca sexta [A56596]), fungi(30% to Trichoderma atroviride [AF188921]), archaea(31% to Thermococcus kodakaraensis [AF188921]), andvertebrates (30% to Homo sapiens [AF290003]). Thehomology search using the BLASTP program did notretrieve plant chitinases and viral chitinases except forthose of baculoviruses.

One of the structural features of chitinases is theirmultidomain structure. The domain structure of the pro-tein also seemed to be identical to that of S. marcescenschiA and AcNPV chitinase (Fig. 2B). The protein motifsearch and sequence alignment indicated that they con-sisted of a signal peptide, module w1, which has beenfound only in the chitinases of some bacteria and baculo-viruses (Perrakis et al., 1994; Henrissat, 1999), and acatalytic domain. In contrast, they shared only catalyticdomains with other chitinases and lacked other domainsor modules, such as a chitin-binding domain, a fibronec-tin type III-like domain, a cellulose-binding domain, andthe Pro/Glu/Ser/Thr-rich (PEST) region, which werefound in other chitinases (Henrissat, 1999).

These features indicate that the protein belongs tofamily 18 chitinases. Moreover, the protein shares exten-sive similarities with S. marcescens chiA and the chitin-ases of baculoviruses in both their amino acid sequencesand domain structures. We designated this novel chitin-ase gene as BmChi-h.

3.4. Phylogenetic analysis

To investigate the evolutionary relationship betweenBmChi-h and other chitinases, phylogenetic analysis was

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e�11

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e�86

r]e�

Fig. 1. Nucleotide and deduced amino acid sequence of the cDNA.The numbers on the left indicate the position from a putative trans-lation start site (+1). (A) Alignment of the nucleotide sequence of the5�-UTR of the cDNA. Note that they are different only in their 5�-UTR but identical to one another in their coding region and –8 to –1bp upstream of the translation start site. (B) Nucleotide and deducedamino acid sequence of the coding region and 3�-UTR of the cDNA.A putative signal peptide is underlined. A putative polyadenylation siteis double-underlined. A putative active site is boxed.

performed. We searched for proteins that show hom-ology to BmCHI-h from public databases, such as Gen-Bank and EMBL, with the BLAST program using thetotal amino acid sequence of BmCHI-h as the query. TheBLAST search retrieved a large number of bacterial andbaculovirus chitinases with extremely low expectancescores (E - value = 0.0) and high sequence homology.In contrast, although chitinases of archaea and eukary-otes were also retrieved, their sequence similarities toBmCHI-h were much lower and limited to their cata-lytic domains.

Proteins used for phylogenetic analysis were mainlyextracted from the SwissProt database (14 proteins). Inaddition, three bacterial chitinases, one baculovirus chi-tinase, and two insect chitinases were selected for analy-sis. Since enzymes belonging to family 18 glycosylhydrolases show homology only in their catalyticdomains (Perrakis et al., 1993; Henrissat, 1999), theamino acid sequences of catalytic domains wereextracted and aligned with the ClustalX program(Thompson et al., 1997), and a phylogenetic tree wasconstructed with the neighbor-joining method (Fig. 3).The phylogenetic tree indicated a general relationship

Fig. 2. Sequence comparisons of BmChi-h, S. marcescens ChiA(SwissProt: P07254), and AcNPV Chitinase (SwissProt: P41684). (A)Total amino acid sequences are aligned with ClustalX program. Aminoacid residues conserved among more than two sequences are high-lighted. The putative active site is underlined. (B) Schematic represen-tation of the domain structure of S. marcescens chiA, AcNPV chiA,and BmCHI-h. The general structure of lepidopteran chitinases isshown for comparison.

between BmCHI-h and chitinolytic enzymes of bacteria,viruses, fungi, and animals. Its topology around S. mar-cescens chiA and the chitinases of baculoviruses wasconsistent with the results of Hawtin et al. (1995).BmCHI-h and baculovirus chitinases were shown tobelong to bacterial lineages with a strong bootstrap sup-port, indicating that BmChi-h had diverged from the chi-tinase gene of the ancestor of a Serratia-like bacteriumor a baculovirus. This evolutionary relationship andstructural similarity between BmCHI-h and the chitinaseof bacteria and baculoviruses suggested the occurrenceof horizontal gene transfer.

3.5. Genomic Southern hybridization and linkageanalysis of BmChi-h

In order to rule out the possibility that BmChi-h wasobtained through contamination with the microbe,Southern blot analysis and linkage analysis were carriedout (Fig. 4).

Fig. 3. Phylogenetic analysis of BmChi-h. Twenty-one chitinaseswere included for analysis. Amino acid sequences of their catalyticdomain were extracted and aligned with the ClustalX program, andpairwise distances were calculated based on the blosum matrix. Thephylogenetic tree was constructed by the neighbor-joining methodusing the MEGA2 program package. Bootstrap values after 1000 repli-cations are shown. Sequences used for analysis: Autographa califor-nica nuclear polyhedrosis virus (SwissProt: P41684), Orgyiapseudotsugata multicapsid polyhedrosis virus (SwissProt: O10363),Xestia c-nigrum granulovirus (GenBank: AF162221), Serratia marces-cens ChiA (SwissProt: P07254), Enterobacter agglomerans (GenBank:U59304), Aeromonas hydrophila (GenBank: AF251793), Alteromonassp. (SwissProt: P32823), Vibrio cholerae (PIR: D82510), Streptomycesplicatus Chi63 (SwissProt: P11220), Bacillus circulans ChiA1(SwissProt: P20533), S. marcescens ChiB (SwissProt: P11797), Cocci-dioides immitis (SwissProt: P54196), Aphanocladium album(SwissProt: P32470), Trichoderma harzianum (SwissProt: P48827),Caenorhabditis elegans CHT-1 (SwissProt: Q11174), Brugia malayi(SwissProt: P29030), Mus musculus CHI3L3 (SwissProt: Q61362),Aedes aegypti (GenBank: AF026491), M. sexta (SwissProt: P36362),and B. mori (GenBank: AB048355).

The copy number of BmChi-h was estimated bySouthern blot analysis. The genomic DNA of the B. morip50T strain was digested with restriction enzymes andprobed with an 854 bp fragment of BmChi-h cDNAamplified by PCR from nucleotide 652 to 1505 bp down-stream of the translation start site. A single band wasdetected in each digest, indicating that BmChi-h was asingle-copy gene. We next performed the linkage analy-sis of BmChi-h. To determine the chromosomal localiz-ation of the BmChi-h gene, we obtained an F1 hybridbetween the B. mori p50T strain and B. mandarina andbackcrossed the F1 hybrid to the p50T strain. The resultof the PCR–RFLP analysis of BmChi-h was identical tothat of Rcf47, a DNA marker of chromosome 7(Sugasaki, personal communication). These results indi-cate that BmChi-h is a single-copy gene and is locatedon chromosome 7 of B. mori. Therefore, we can rule

Fig. 4. Southern blot analysis of BmChi-h. (A) Genomic DNA of B.mori strain p50T (10 µg) and BAC DNA of clone 93-16L (2 µg) weredigested and blotted onto a positively charged nylon membrane andhybridized with a 32P-labeled probe. The position of the probe is shownin Fig. 6. Position and sizes of molecular markers are indicated to theleft of the figure. The numbers shown above the figure indicate therestriction enzymes that digest genomic DNA or BAC DNA (lane 1:EcoRI, 2: EcoRV, 3: StuI, 4: ScaI, 5: SacI, 6: PstI). (B) Linkage map-ping of BmChi-h using PCR–restriction fragment length polymorphism(PCR-RFLP). Thirty individuals of (p50T × B. mandarina) × p50Twere examined. Figures indicate the number of individuals that arehomozygotes for the p50T-type alleles or heterozygotes for the p50T-type and B. mandarina-type alleles.

out the possibility that BmChi-h was obtained throughcontamination of the microbe.

3.6. Northern blot analysis

The expression profile of BmChi-h mRNA was exam-ined by Northern blot analysis and compared with thatof the B. mori chitinase gene reported previously (Kogaet al., 1997; Kim et al., 1998; Mikitani et al., 2000;Abdel-Banat and Koga, 2001) (Fig. 5). In the epidermisand midgut, strong signals of 2.4 kb, which is consistentwith the length of cDNA, were observed on day 5 of the

Fig. 5. Expression profile of BmChi-h mRNA from fourth instar lar-vae to day-1 pupae. Total RNA (10 µg) extracted from epidermis,midgut, and fat body were blotted onto a positively charged nylonmembrane and hybridized with the same probe used for Southern blotanalysis. The same Northern blot was rehybridized successively withprobes for B. mori Chitinase and B. mori Actin3. Actin3 is shown asa control. Molecular sizes are shown to the right of the figures. Stagesare indicated above the figure.

fourth instar, at the ecdysteroid peak, and decreased tothe basal level by day 2 of the fifth instar. In the fifthinstar, the signal was hardly detected at the feeding stageand appeared again on day 9, just after the start of spin-ning. Its maximum expression was observed on day 11,two days before pupal ecdysis. In the fat body, BmChi-h mRNA was detected only on day 5 of the fourth instarand day 9 of the fifth instar, and the signal was muchweaker compared with that in epidermis and midgut.This stage- and tissue-specific expression profile wasconsistent with the result of another chitinase gene ofB. mori.

3.7. Genomic structure of BmChi-h

The genomic structure of BmChi-h was determined byshotgun sequencing of a BAC clone. The BAC librarythat is assumed to contain 11 genome equivalents of B.mori (Koike et al., 2003) was screened and 15 BACclones were identified to be positive. The nucleotidesequences of 93-16L, one of the positive clones (Fig.4A), were analyzed by shotgun sequencing, and six con-tigs covering about 158 kb in total were constructed fromthe raw sequence data.

The assembled nucleotide sequences revealed theoverall genomic organization of BmChi-h (Fig. 6). There

Fig. 6. Overall genomic structure of the BmChi-h gene. While thereare no introns in the ORF of BmChi-h, there are three isoforms in its5�-UTR, which are generated by different promoter usage and splicing.(A) Schematic representation of the genomic structure of the BmChi-h gene. The BAC clone 93-16L was shotgun-sequenced, and one con-tig (about 15.7 kb) was found to encompass the BmChi-h gene. Nucleo-tide sequences of the 5�-UTR were found about 10 kb, 5 kb, and 50bp upstream of the translation start site, and the mRNA isoforms werenamed BmChi-hA, BmChi-hB, and BmChi-hC, respectively. Numbersindicate the position from translation start site, and the coding regionis shown by arrowheads. The position of the probe used for Southernblot analysis is also shown. (B), (C), and (D) Nucleotide sequencesaround the 5�-end of BmChi-hA, BmChi-hB, and BmChi-hC, respect-ively. The nucleotide sequences specific to each isoform are boxed,and the overlapping sequence among them is double-lined. Arrow-heads indicate the 5�-end of the 5�-RACE product, and numbers indi-cate their abundance. The most abundant position was determined asa transcription start site. The consensus splicing sequences are indi-cated by broken lines. The coding region is shown in bold. The puta-tive TATA box is shown in italics. Sequences similar to the arthropodcapsite (Cherbas and Cherbas, 1993) are underlined.

was no intron in the coding region of BmChi-h. How-ever, there seemed to be introns in the 5�-UTR. 5�-RACEindicated that there were three isoforms in the 5�-UTRof BmChi-h (Fig. 1). Nucleotide sequences identical tothe 5�-UTR of each isoform were found �10 304 to

�10 258, �5085 to �4944, and �56 to �9 bp upstreamof the translation start site. The 5�-UTR sequences over-lapping among the three isoforms were found �8 to �1bp upstream of the translation start site. The consensussplicing donor sites that can construct these three mRNAisoforms were found �10 257 to �10 252 bp(GTGGGT; Fig. 6B) and �4943 to �4938 bp(GTAAGT; Fig. 6C) upstream of the translation startsite, and the consensus splicing acceptor site was foundjust upstream of the overlapping region(GCTTTTTTGTAG; �20 to �9 bp; Fig. 6D). A puta-tive TATA box sequence was present just upstream ofthe 5�-end of the type C isoform (�92 to �86 bpupstream of the translation start site). The sequencessimilar to arthropod capsite consensus sequences(Cherbas and Cherbas, 1993) were found near the 5�-ends of all three isoforms. The similar sequences ofecdysteroid-responsive elements, RGG/TTCANTGAC/ACY (Cherbas et al., 1991), were found �11 023 to�11 011 bp (GGTTCAGTGAGAT), �8756 to �8744bp (ATTTCAATGACGT), and �8653 to �8641 bp(AGTTCACTGAATT) upstream of the translationstart site.

These results indicated that the three isoforms in 5�-UTR were generated by alternative use of three differentpromoters. The existence of the similar sequences ofecdysteroid-responsive elements in the 5�-flankingregion, together with the fact that BmChi-h mRNA wasobserved only at molting stages, suggested that tran-scription of BmChi-h was controlled by the ecdysteroid-dependent regulation mechanism.

4. Discussion

In this study, we cloned and characterized a novel chi-tinase gene from B. mori. We searched the EST databaseof B. mori and found that there were five non-redundantcDNAs encoding chitinases or chitinase-like proteins inSilkBase (Table 1). Although it remains to be clarifiedwhether they are derived from true transcripts of B. mori,it is probable that chitinases are a multigene family inB. mori, as they are in dipteran insects (de la Vega etal., 1998).

It was a surprising result that BmChi-h cDNA encodeda protein that shared extreme similarities with bacterialand baculovirus chitinases in both their amino acidsequences and domain architectures (Fig. 2). We firstdoubted contamination with microbes or viruses; how-ever, we could prove that BmChi-h was a genuine geneof B. mori and that it was located on chromosome 7(Fig. 3).

The expression of BmChi-h mRNA was observed ina stage- and tissue-specific manner (Fig. 5). Strongexpressions during the molting stages indicated that thetranscription of BmChi-h was induced only at the molt-

ing stages, probably by the presence of ecdysteroids.Strong expressions in epidermis and midgut and weakexpression in fat body suggested that BmChi-h wasstrongly induced in chitin-containing tissues. This stage-and tissue-specific expression profile was almost ident-ical to that of another chitinase of B. mori, indicatingthat it was controlled by a similar mechanism. Wedetermined the overall genome structure of BmChi-h byshotgun sequencing analysis of the BAC clone and foundthat BmChi-h had three promoters that generated threeisoforms in their 5�-UTR (Fig. 6), although the charac-teristics of each isoform should be examined in a furtherstudy. Since chitinases are believed to be regulated byecdysteroid hormones in lepidopteran insects (Kramer etal., 1993), these two chitinase genes would share com-mon cis-elements, such as ecdysteroid-responsiveelements (Cherbas et al., 1991). Actually, we were ableto find the similar sequences of ecdysteroid-responsiveelements in the 5�-flanking regions of BmChi-h (Fig. 6).Our study would facilitate further study of the molecularmechanism of tissue-specific and hormone-dependentexpression of the chitinase gene.

The isolation of a chitinase gene showing high hom-ology to bacterial and baculovirus chitinases from a lepi-dopteran insect has great significance from the evolutionaspect as well, because it has been proposed that anancestral baculovirus acquired the chitinase gene from aSerratia-like bacterium via horizontal gene transfer(Hawtin et al., 1995; Kang et al., 1998). We speculatethat the origin of the BmChi-h gene is the chitinase geneof a Serratia-like bacterium or an ancestral baculovirusbased on the following reasons, in addition to their struc-tural similarities (Fig. 2). First, phylogenetic analysisshowed that BmChi-h and baculovirus chitinasesbelonged to bacterial lineages with strong bootstrap sup-port (Fig. 3), suggesting that they have evolved from thechitinase gene of a bacterium. Second, the distributionof orthologous genes of S. marcescens chiA seems to bequite limited. So far, orthologous genes of BmChi-h havenot been found in any eukaryotes, archaea, or viruses,including organisms whose genomes have beensequenced, except for B. mori and baculoviruses.Although homology search in public databases retrievedthe sequences from eukaryotes and archaea, their hom-ology was much lower and limited to their catalyticdomains. Third, baculovirus and S. marcescens are eco-logically intimate to lepidopteran insects. Baculovirusesform a large group of dsDNA viruses that infect mainlylepidopteran insects. S. marcescens is an enteric patho-gen of a wide variety of animals, and it is often foundin insect gut. Baculoviruses that infect insects replicatetheir genome within the nucleus of their host, and S.marcescens can invade the insect body cavity throughmidgut cells. There may be opportunity for a baculovirusand a Serratia-like entomopathogenic bacterium to haveexposure to host DNA. Fourth, accumulating data sug-

gest that baculoviruses have evolved through capturinggenes and transposable elements from their host insects(Miller and Miller, 1982; Lerch and Friesen, 1992; Jehleet al., 1998; Handler et al., 2002). It is now supposedthat baculoviruses can spontaneously accommodatetransposable elements from their host and might transferthem to a new host or other co-infecting viruses (Fraser,1986; Jehleet al., 1998; Handler et al., 2002). In addition,some reports suggest that horizontal gene transfersbetween insect viruses and their insect hosts are not lim-ited to transposable elements but also occur in the caseof functional genes (Becker, 2000; Chen et al., 2001;Pearson and Rohrmann, 2002). Some genes of baculo-viruses are known to show extensive similarities to thegenes of host insects (O’Reilly and Miller, 1989; Guar-ino, 1990; Hawtin et al., 1995; Becker, 2000).

If this hypothesis is true, several issues need to beaddressed. The first issue is the mechanism and directionby which the chitinase gene was transferred horizontally.One possibility is that a virus captured the gene from abacterium and then transferred it to an insect genome.The second is that the gene was first transferred to aninsect genome from a bacterium and then a virus cap-tured the gene from an insect. The third is that the bac-terial chitinase gene was transferred independently toviral and insect genome. We determined the genomestructure of about 158 kb around BmChi-h with theexpectation of evidence of horizontal gene transfer.However, there was only a BmChi-h gene in this region,and we could not find traces of gene transfer, such asanomalous nucleotide composition and bacteria- orvirus-derived sequences (data not shown). The secondissue that needs to be addressed is whether there is analternative explanation of gene losses in multiple lin-eages. Although orthologous genes of BmChi-h did notseem to exist in the genome of D. melanogaster andAnopheles gambiae, whose genomes have beensequenced fully, there still remains the possibility thatorthologous genes of S. marcescens chiA have experi-enced frequent gene losses in most lineages of eukary-otes. To determine whether this hypothesis is correct,we need further investigations, such as a study on thedistribution of orthologous genes in other arthropods.The third issue is how the BmChi-h-like chitinase genecame to be regulated in the genome of a lepidopteraninsect. It is likely that a horizontally transferred genecan be much more easily maintained and will prevailin populations if it confers a selective advantage on therecipient organism (Lawrence, 1999; Koonin et al.,2001). In baculoviruses, the acquisition of chitinase geneis believed to increase pathogenicity (Hawtin et al.,1997; Gooday, 1999). It is probable that BmCHI-h haschitinolytic activity and is involved in the developmentalprocess of chitin degradation at molting stages. BmChi-h was mapped to chromosome 7, which is the same asthat of another chitinase gene (Mikitani et al., 2000) and

fungal resistance gene called cal (Aratake, 1961). Sincethe antifungal activity of some chitinases has been util-ized to produce transgenic plants resistant to pathogenicfungi (Wang et al., 1996), the chitinases of B. mori mightbe associated with fungal resistance. We do not knowthe answer to the third question at this point; however,further study of the enzymatic property of BmCHI-hmight provide a clue to its answer.

Acknowledgements

This work was supported by the Basic Research Pro-gram, BRAIN (to K.M. and M.K.), the Insect GenomeResearch Program, NIAS (to K.M. and T.S.), andGrants-in-Aid for Scientific Research (nos. 14360032and 14656023), JSPS (to T.S.). We are grateful to N.Omuro for technical assistance.

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a bombyx mori gene, bmchi-h, encodes a protein homologous to bacterial and baculovirus chitinases

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