Alternative splicing of the primary transcript generates heterogeneity within the products of the gene for Bombyx mori chitinase.

The gene of chitinase in the silkworm, Bombyx mori, generates four mRNA products by alternative splicing. Nucleotide sequences of the entire gene for chitinase and respective cDNAs demonstrate that the pre-mRNA undergoes alternative splicing at both the 5' and 3' regions. At the 5' region, the pre-mRNA experienced differential splicing through two alternative 5'-intron consensus splicing sites. These products differ in the last amino acid of the signal peptide and the first amino acid of the mature N-terminal sequences: one with Cys(20)-Ala(21) and the other with Ser(20)-Asp(21). The product with Cys(20)-Ala(21) residues is one amino acid larger than the other with Ser(20)-Asp(21). At the 3' region the pre-mRNA of the chitinase gene undergoes alternative splicing in three different fashions. It is spliced either through retaining or excluding the upstream 121-bp direct repeat found at the 3' region of the coding sequences or through retaining or excluding of an insertion of 9 bp in a combinatorial manner. Retention or exclusion of the upstream 121-bp direct repeat results in a protein with a deduced amino acid sequence similar in size to the one retaining both direct repeats. However, exclusion of the insert of the 9 bp from the mRNA results in a protein with 22 extra amino acids. All of the mRNA products appear to be generated from a single gene as demonstrated by testing the 3' region of the genomic DNA and variant chitinase mRNA products. B. mori chitinase expression in the fifth instar larvae epidermal tissues appears to be developmentally regulated, but the phenomenon of alternative splicing of the pre-mRNA is not stage-dependent. Furthermore, the four mRNA products showed chitinase activity when expressed in Escherichia coli, which demonstrates the role of the alternative splicing process in generating multiple isoforms of the silkworm's chitinase.

There are many sources of chitinases, including bacteria, fungi, plants, marine organisms, insects, and mammals (1). The role of these enzymes differs among the organisms. In insects, they degrade the biopolymer chitin, which is found in the exoskeleton and gut lining. Insect chitinases are induced by ecdysteroids at the time of molting and metamorphosis of the larvae to degrade most of the older chitin (2,3). Chitinase expression in the insects' exoskeletons and the guts normally occurs only during molting, where the chitin of the integument and the peritrophic membrane is presumably degraded (4). Expression of the chitinase at a precise timing in insects demonstrates its fundamental role in the process of ecdysis. To elucidate the functions of the chitinases, the genes for these enzymes have also been isolated and characterized from many organisms, including mammals, insects, fungi, nematodes, and bacteria (5)(6)(7)(8)(9)(10)(11)(12). One of the structural features observed in chitinases is a multidomain architecture that includes catalytic domains, a cysteine rich chitin-binding domain, and a serine/ threonine-rich domain that is glycosylated (13). The conserved motifs in most of the insects' chitinase genes reside in the central domains, which include the two regions implicated in catalysis (9). Differences occur in the cysteine-rich carboxyl domain and the proline/glutamate/serine/threonine-rich domain that is presumed to increase the susceptibility to proteolysis by a calcium-dependent protease (14,15). The silkworm, Bombyx mori, chitinases are well studied in our laboratory at both the enzymological and DNA levels (9,16,17).
We have previously reported a complete exploration of the gene for the chitinase from B. mori (9). The gene's structural organization and functional motifs are consistent with the multidomain architecture of the proteins (13). The organizations of its functional domains are identical to the related gene from Manduca sexta (18). Hence, the genes for cuticular chitinases may be conserved among insect species (9). The coding regions of the genomic clone for the gene of B. mori chitinase have unique features both at exon 1/intron 1 splicing junctions and the 3Ј-end of the gene's coding regions (Fig. 1). First, the invariant nucleotides, GT, at the 5Ј boundary of intron 1 are repeated in near proximity, so we suggested a differential splicing of the pre-mRNA (9). Another feature of this gene includes the incorporation of an additional sequence of 9 bp at the 3Ј-end of the coding regions (Fig. 1). This sequence is absent from a chitinase cDNA clone studied earlier (7). The open reading frame (ORF) 1 is interrupted by the inclusion of the 9 bp due to the stop codon within these nucleotides. Therefore, the protein encoded by our chitinase gene is 22 amino acids smaller in size. Furthermore, another cDNA encoding a putative chitinase of B. mori has been reported recently (8). There are some discrepancies between these two sequences of the chitinase cDNAs (7,8). They differ in the first amino acid of the mature N-terminal region and in the C-terminal coding region. In addition, there are direct repeats of 112 bp in the former cDNA sequence (7), but these were not found in the latter study (8). Our previous studies on B. mori chitinases revealed that the first amino acid of the mature N-terminal sequences is alanine (16). These results are supported by deduced amino acid sequences of the chitinase cDNA that was studied by Mikitani et al. (8). Our study of the genomic clone for the gene of chitinase suggested the possible occurrence of all cDNAs sequences in a condition where an alternative splicing of the primary transcript of the chitinase occurs in the cell's nucleus.
Alternative pre-mRNA splicing is an important mechanism in gene regulation and can include the use of alternative 5Јand/or 3Ј-splice sites, exon skipping, mutual exon exclusion, and/or intron retention (19 -21). Although intron retention is common in viral pre-mRNAs, it appears to be a relatively rare form of alternative splicing in vertebrates (22). It is believed that intron-containing mRNAs normally are prevented from being transported to the cytoplasm due to the formation of the spliceosome complex, which commits the pre-mRNA to the splicing pathway (23)(24)(25)(26). However, there are examples of alternative intron retention in vertebrates that result in novel proteins due to translation of the intron sequences (27)(28)(29)(30). Therefore, there must be a mechanism by which a fraction of the mRNA molecules, containing an intact intron, escape the splicing pathway (20). Because both the 5Ј-and 3Ј-splice sites are required for commitment of spliceosome complex formation (31), it is possible that some suboptimal splice sites may prevent complex formation, thereby allowing an intron to be retained and intron-containing mRNAs to be transported to the cytoplasm.
To obtain more insight into the splicing mechanism of chitinase primary transcript and to clarify the discrepancies observed on the N-and C-terminal amino acids, we have cloned B. mori chitinase cDNAs from different stages of the fifth instar larvae and investigated the role of alternative pre-mRNA splicing in generating heterogeneous chitinase cDNAs within the silkworm. The results of this study are here described and discussed.

EXPERIMENTAL PROCEDURES
Preparation of Total RNA from B. mori Cuticular Tissues-The silkworms, B. mori strain (Kinshu ϫ Showa), were reared on mulberry leaves at a room temperature of 27°C with a photoperiod of 13 h:11 h light/dark. The epidermal tissues of the integument were collected from the fifth instar larvae on the day when spinning behavior started (SP-0) and 1, 2, 2.5, and 3 days later (SP-1, SP-2, SP-2.5, and SP-3, respectively). The tissues were prepared as described previously (9). Total RNAs were isolated from the frozen tissues using the RNeasy Midi kit (Qiagen, Inc.) according to its protocol.
Chitinase cDNA Synthesis and Amplification-Chitinase cDNAs were synthesized using the total RNAs obtained from the five different stages with the Ready-To-Go RT-PCR kit (Amersham Biosciences) according to the instructions of the manufacturer. The first-strand cDNA synthesis was done on 2 g of total RNA using reverse transcriptase (FPLCpure) and the oligo(dT) primer, pd(T) 12-18 (Amersham Biosciences). To amplify the cDNAs, each PCR was primed with two gene-specific primers designed from the B. mori genomic clone for the chitinase gene (9). The primers used in these amplifications were: a sense primer designed at the 5Ј-end of the gene (BMB XbaI 1 (5Ј-GC-GTCTAGAGAAAATGCGAGCGATATTTG-3Ј), the underlined sequence is the site for XbaI] and three antisense primers. One of them starts from the 9-bp insert DNA, which is found in the genomic clone, and contains a stop codon. Its position is at nucleotides 13,828 -13,847 of the genomic clone for the chitinase gene (9) (BMB SacI 8 (5Ј-GCGCGAGC-TCTTACGAACATTCCGGTCTG-3Ј)). The second antisense primer was designed at nucleotide positions 14,534 -14,553 of the genomic clone (9) and named BMB SacI 10 (5Ј-GCGCGAGCTCGGGTCGACGTAAACAT-TGG-3Ј). The underlined sequences in both primers represent the SacI recognition site designed within the primers. The third antisense primer was synthesized at the 3Ј-end of the gene with the XhoI recognition site (BMb14 (5Ј-GCGCTCGAGCCTTCTGCTCTCATTTG-3Ј)). Amplification reactions (50 l) contained 7 l of first-strand cDNAs, 5 l of 10ϫ Ex Taq buffer, 4 l of dNTP mixture (2.5 mM each), 1 l of sense and antisense primers (20 pmol of each), 31.8 l of sterile distilled water, and 0.2 l of TaKaRa Ex Taq DNA polymerase (5 units/l) (Takara Shuzo Co., Ltd.). Cycling parameters were set as follows: one cycle of preheating at 94°C for 3 min and 30 -40 cycles of each of denaturation at 94°C for 30 s, annealing at 60°C for 1 min, and ex- The alternate 5Ј-intron 1 splicing site is indicated by the shill mark (/). The underlined nucleotides code for the underlined amino acid, cysteine, while the boxed nucleotides code for the boxed amino acid, alanine. The sequences of the 121-bp direct repeats in exon 10 that are indicated by white arrows within the structural scheme are underscored with inverted arrows, and the 9-bp insert DNA is boxed. The stop codon, TAA, is in boldface letters. tension at 72°C for 3 min. A final post-extension cycle was performed at 72°C for 7 min.
Southern Blotting and Hybridization-A cDNA clone of 1758 base pairs in length previously synthesized and detected containing an insert of 9 bp by sequence analysis was used as probe. It was prepared from the SP-3 stage of the fifth instar larvae using the sense primer, BMB XbaI 1, and an antisense primer, BM5 (5Ј-GGCGAATTCCTTAC-GAACATTCCGGTCT-3Ј), which was designed from the genomic DNA sequence (9) at nucleotide positions 13,950 -13,968. The probe was labeled using the Gene Images Random Prime Labeling Module (Amersham Biosciences). Electrophoresis of the target DNAs and their transfer onto the membrane was carried out as described previously (9). The DNAs were allowed to transfer overnight onto the Hybond-N ϩ membrane and then fixed to the membrane by heating at 80°C for 2 h.
Hybridization buffer was composed of 5ϫ SSC containing 0.1% SDS, 5% dextran sulfate, and 20-fold dilution of liquid block. Hybridization was performed overnight at 65°C with gentle agitation. The blots were washed for 15 min in the first stringency wash solution (1ϫ SSC containing 0.1% SDS), which was pre-heated to 65°C. A further stringency wash was carried out at the same temperature in a pre-heated solution of 0.5ϫ SSC containing 0.1% SDS. Blocking of the blots, antibody reaction, removal of the unbound conjugate, signal generation, and detection were performed according to the manufacturer's protocol. 10-min exposures were made of the membrane using Fuji x-ray film, and the film was developed.
Expression of Chitinase in Fifth Instar Larvae-Total RNA was extracted from five stages of the fifth instar larvae as described above. For Northern hybridization analysis, the total RNAs (10 g from each of SP-0, SP-1, SP-2, SP-2.5, and SP-3) were denatured in formaldehyde, electrophoresed in a denaturing 1.2% agarose gel system, transferred to a Hybond-N ϩ membrane by the capillary method, and immobilized by laying the membrane for 5 min on Whatman 3MM paper, which had been soaked in 0.4 M NaOH and briefly rinsed in 2ϫ SSC to remove the NaOH after fixing. Hybridization of the RNAs was carried out at 65°C overnight with a cDNA probe amplified with the primers BMB XbaI 1 and BMB SacI 10 and labeled in the same manner as described under "Southern Blotting and Hybridization." The first stringency wash was performed at 65°C for 15 min in 1ϫ SSC containing 0.1% SDS, whereas the second wash was carried out at the same temperature for 15 min in a solution of 0.1ϫ SSC containing 0.1% SDS. Blocking of the blots, antibody reaction, removal of the unbound conjugate, signal generation, and detection were performed according to the manufacturer's protocol. 5-h exposures of the membrane were made to Fuji x-ray film, and the film was developed.
cDNA Cloning and DNA Sequencing-The amplified cDNAs were either treated with SacI, XbaI, and XhoI restriction endonucleases and cloned into pBluescript II SKϩ vector (Stratagene) or directly cloned into pCR4-TOPO vector (Invitrogen) and sequenced by a modified Sanger's dideoxynucleotide-mediated chain termination method using the Thermo Sequenase Cycle Sequencing kit (Amersham Biosciences) and the M13 reverse and M13 IRD800 infrared dye-labeled primers. Further sequence analysis operations, such as comparison, restriction and translation, were conducted with Mac Molly software for Macintosh computers. Multiple sequence alignments were accomplished using the program GENETYX-MAC 7.2 #1.
Expression of the Recombinant Chitinases in Escherichia coli-Four cDNAs corresponding to the clones SP-1/(1-10)1, SP-2/(1-10)2, SP-2/ (1-10)1, and SP-2.5/(1-8) (Table II) were amplified by PCR using the primers: sense, 5Ј-GCGAGCGATATTTGCGACGTTGGCTGTCC-3Ј, which starts priming from the nucleotide G of the initiation codon, ATG, of the chitinase cDNAs; antisense, 5Ј-CCCCGAATTCGGGTCGACGTA-AACATTGG-3Ј (for amplification of the first three clones) and 5Ј-GGC-GAATTCCTTACGAACATTCCGGTCT-3Ј (for amplification of the last clone) (EcoRI site underlined), and the thermostable polymerase possessing a proofreading activity, Elongase Enzyme Mix (Invitrogen). These amplified cDNAs were inserted into the EcoRI and EcoRV cloning sites of the pETBlue-1 expression vector (Novagen) and transformed into the cloning host, NovaBlue competent cells, using standard protocols. The presence of the inserts and their orientations were determined using direct colony PCR with a vector-specific primer and an insertspecific primer. The newly constructed plasmids were called pETBlue-1-SP-1/(1-10)1, pETBlue-1-SP-2/(1-10)2, pETBlue-1-SP-2/(1-10)1, and pETBlue-1-SP-2.5/(1-8). E. coli strain Tuner(DE3)pLacI (Novagen) was transformed with the plasmids described above and grown in 3 ml of LB medium supplemented with 1% glucose, 50 g/ml carbenicillin, and 34 g/ml chloramphenicol at 37°C to absorbance of 0.5 at 600 nm. The cultures were diluted 1:50 and grown at 37°C in the same LB medium until the absorbance at 600 nm is ϳ0.6 -1.0. Expression of the chiti-nases was induced by addition of isopropyl ␤-D-thiogalactopyranoside (IPTG) to a final concentration of 0.6 mM. The cultures were shifted to 30°C for 4 h. The cells were harvested from the liquid cultures by centrifugation at 6500 ϫ g for 15 min at 4°C. The cell pellets were suspended in 20 mM Tris-HCl (pH 7.5) containing 10 mM EDTA and 1% Triton X-100. Cell lysis was done by sonication and lysozyme treatment, and the inclusion bodies were collected by centrifugation at 10,000 ϫ g for 10 min. The inclusion bodies were suspended at a concentration of 20 mg/ml in a solubilization buffer, 50 mM CAPS (pH 11.0) supplemented with 0.3% N-lauroylsarcosine and 1 mM dithiothreitol (DTT), and then incubated at room temperature for 15 min. The solubilized proteins were clarified by centrifugation at 10,000 ϫ g for 10 min at room temperature. The soluble fractions were dialyzed twice against 20 mM Tris-HCl (pH 8.5) containing 0.1 mM DTT, each for 3 h at 4°C. Another two dialysis processes were done using the same dialysis buffer but lacking DTT.
SDS-PAGE and Staining-SDS-PAGE was done with a 10% polyacrylamide slab gel containing 0.1% SDS. Total cell proteins from the induced or uninduced bacterial cells were mixed with 3ϫ SDS-PAGE sample loading buffer, 0.3 M Tris-HCl buffer (pH 6.8) containing 3% SDS, 30 mM DTT, 30% glycerol, and 3 ϫ 10 Ϫ3 % bromphenol blue. The  (1-14). The deduced amino acid sequence is shown on the top of the nucleotide sequence. The upward arrowhead indicates the position of the differentially spliced triplet (GTG). The 9-bp insert DNA is boxed, and the stop codon within the insert, TAA, is shown in boldface letters. The sequences of the direct repeats are underscored with dotted lines with arrowheads at the ends. The putative chitinase signal peptide sequence is underlined. The first residue of the mature N-terminal amino acids, aspartate, is also boxed. The sequence has been deposited in the GenBank TM data base (accession number AB052914). Native-PAGE and Activity Staining-Activity staining for chitinase was done by the method of Koga et al. (32). The proteins were first subjected to a 7.5% polyacrylamide gel electrophoresis under nondenaturing conditions and then transblotted to 7.5% polyacrylamide slab gel containing 0.01% glycol chitin at 90 mA for 20 min in 5 mM Tris-glycine buffer (pH 8.3). The transblotted gel was incubated in 100 ml of 100 mM sodium phosphate buffer (pH 8.0) for 2 h at room temperature. The gel was soaked in 100 ml of 100 mM Tris-HCl (pH 9.0) containing a chitinadsorbing brightener, 0.01% Fluostain I (Dojin Chemical Laboratory) for 5 min, rinsed with distilled water to remove the brightener, and then immersed into 100 mM sodium phosphate buffer (pH 8.0). The chitinase activity bands were detected by irradiation with ultraviolet light.

RESULTS
Isolation and Amplification of Chitinase cDNA Clones from SP-3 Larvae-The total RNA was extracted from fifth instar larval integuments 3 days after the larvae started cocoonspinning behavior (SP-3). During these days the larvae had not yet undergone apolysis, because around the time of apolysis the chitinolytic enzyme activity is maximal (6,16,33) and the mRNA is expected to be translated into chitinase. The mRNA content in the SP-3 total RNA was converted into the firststrand cDNA by reverse transcriptase using the oligo(dT) primer, pd(T) [12][13][14][15][16][17][18] (Amersham Biosciences). The first-strand cDNA was used as template for PCR amplification. To obtain diverse B. mori chitinase cDNAs, three different sizes of cDNAs were amplified with the gene-specific primers designed from the sequence of the genomic clone for the chitinase gene (9) (Fig. 2A). The sizes of the fragments obtained were 1637, 2346, and 2791 bp. The smallest fragment ends with the 9-bp insertion, and the longest one ends downstream of the putative polyadenylation signal (Fig. 2A). These fragments were subcloned separately into the pBluescript vector and transformed into E. coli. The cDNAs obtained from individual bacterial colonies that were transformed with the longest fragment (2791 bp) were subjected to Southern blotting using a cDNA clone whose sequence was identified earlier as a probe (Fig.  2B). The other subcloned fragments (1637 and 2346 bp) were tested only by PCR amplifications, because a common sense primer was used for all PCRs, and therefore the amplified products were expected to be from the same target gene. The recombinant pBluescripts with the different clones from the SP-3 larvae were sequenced, and the results from six sequencing rounds revealed that all clones sequenced so far with homologous sequences differed only in their corresponding sizes (Fig. 3). Interestingly, all cDNAs sequenced carried the insert of 9 bp at the same position, which includes a stop codon, TAA. This 9-bp insertion mediates a direct repeated sequence of 112 bp. If the nucleotides of this insertion were summed up to the upstream 112 nucleotides, the direct repeats will be of 121 nucleotides (Fig. 1). The complete sequence of the cDNA had an ORF of 1629 nucleotides, encoding a protein of 543 amino acid residues (Fig. 3) that has a calculated molecular mass of 60,979 Da.
Identification of Chitinase cDNA Clones Generated by Alternative Splicing of Pre-mRNA-Analysis of the B. mori genomic clone for the chitinase gene (9) brought to light the possibility of alternative splicing of the pre-mRNA to yield variant mature mRNAs (Table I and Fig. 1). To prove the occurrence of this phenomenon in B. mori chitinase gene, many other cDNAs of different sizes were synthesized and analyzed from the stages SP-0 through SP-2.5 of the fifth instar larvae. Sequence analysis of the cDNA clones revealed that alternative splicing of the primary transcript yielded variant mature mRNAs of the B. mori chitinase gene (Figs. 3 and 4). The relative distributions of the products of the alternatively spliced mRNAs are summarized in Table II (1-14)) were lacking these nucleotides. The mRNAs of the six clones were found to retain the 9 bp at the 3Ј-end of the gene, whereas those of the other two clones excluded these nucleotides. Furthermore, in three clones (SP-1/(1-10)2, SP-2/(1-10)2, and SP-2/ (1-14)), the upstream 121-bp direct repeat, including the 9 bp, underwent alternative splicing to become part of intron number 9 of the chitinase gene (Table II and Fig. 5, A and B). Retention and exclusion of these sequences appears to be independent of the stage of the fifth instar (Table II).  Effect of Alternative Splicing on Generating Heterogeneity within the Gene Products for Chitinase-To investigate the effect of alternative splicing on generating variations within the products of the primary transcript of B. mori chitinase gene, the deduced amino acid sequences of B. mori chitinase cDNAs elaborated from the sequenced clones were aligned (Fig.  6). Sequence alignment revealed that the encoded chitinase from the different clones showed exactly the same amino acid residues. However, retention of the triplet (GTG) at the exon 1 and intron 1 splicing boundary changes the last amino acid residue of the putative chitinase signal peptide from serine to cysteine and introduces additional amino acid, alanine, at the mature N-terminal sequence. This alanine is consistent with the chitinase N-terminal sequence (ADSRARIVCYFSNWAV-YRPG) (16). Exclusion of the triplet results in a mature Nterminal sequence that starts with aspartate, and the last amino acid of the putative signal peptide is serine. The addi-tional nucleotides of 9 bp at the 3Ј-end of the coding region reduced the size of the encoded protein by 22 amino acids, because they generate a premature stop codon. On the other hand, exclusion of the 9 bp resulted in a primary transcript encoding a protein of larger size (i.e. 566 and 565 amino acids in clones with or without alanine at the N-terminal region of the deduced chitinase sequence, respectively). Exclusion of the upstream 121-bp direct repeat resulted in a protein of similar size when compared with the one retaining the 9 bp. In the former case only the position of the stop codon was shifted to a new position on the downstream 121-bp direct repeat (Fig. 6).
Confirmation of the Role of Pre-mRNA Splicing-To verify that the cDNAs described above were produced by alternative splicing and were not transcribed from additional gene sequences elsewhere in the genome, the same primer set was used in PCR with genomic DNA as the template. The expected size of genomic PCR product for the primer set was obtained. The DNA fragments amplified from SP-0 through SP-3 genomic DNAs were typically the same size as expected (Fig.  7A). The same primer set was used in PCR with cDNA clones that exhibited differential splicing of the upstream 121-bp direct repeat (Fig. 7B). The results indicated that the variant cDNA clones investigated were the products of a single gene, which in turn supported the notion that the copy number of the B. mori chitinase gene was 1 per haploid genome (8). In addition, the results are not consistent with the presence of a large intron separating the repeated sequences.
Developmental Changes in Chitinase mRNA Levels-Chitinase cDNAs were synthesized from the fifth instar larvae on the day of spinning behavior through 3 days later (SP-0 through SP-3). To assess the changes in the levels of chitinase mRNA in these epidermal tissues, total RNAs from the respective tissues were analyzed by Northern blotting. The blots were probed with the fragment BMB XbaI 1 ϳ BMB SacI 10 ( Fig.  2A). Chitinase mRNA was undetected by Northern blotting that only the 9-bp insert was spliced in a combinatorial fashion. B, the top diagram is similar to that in A, illustrating that the 9-bp and the upstream 112-bp direct repeat were spliced as part of intron 9. The product of the primary transcript is shown at the bottom. C, the structure of a previously reported cDNA clone (8). The clone retained the triplet (GTG) at the 5Ј region as shown by ϩAla 21 but excluded the upstream 121-bp direct repeat. D, the structure of another chitinase cDNA clone (7). This clone excluded both the triplet (GTG) at the 5Ј region as indicated by ϪAla 21 and the 9-bp insert, which is at the end of the upstream 121-bp direct repeat. However, the clone retained the 112-bp direct repeats as shown by 112 and 121 bp below the diagram.
ϪGTG Ϫ9 a ϪGTG and ϩGTG indicate the cDNA clones without (Ϫ) or with (ϩ) the GTG triplet at the 5Ј-region. ϩ9 and Ϫ9 indicate the cDNA clones with (ϩ) or without (Ϫ) the 9-bp insert DNA at the end of the upstream 121-bp direct repeat. Ϫ121 represents exclusion of the upstream 112-bp direct repeat together with the 9-bp insert DNA from the cDNA clone. on the day when fifth instar larvae initiated spinning behavior. However, it was present at very low levels 1 day later, and its levels increased sharply on the second day and gradually onward (Fig. 8A). To test the level of the expressed chitinase mRNA that affect detection of the mature chitinase transcripts by RT-PCR, cDNAs were synthesized using half the amount (5 g) of the total RNAs used for Northern blotting as templates for the first-strand cDNA synthesis. As shown in Fig. 8B, all cDNAs plotted were amplified with the B. mori chitinase gene-specific primers, BMB XbaI 1 and BMB SacI 10, and the respective first-strand reactions as templates for PCR. The results showed expression of the chitinase mRNAs in all stages of the fifth instar larvae investigated, which means the chitinase in B. mori is gradually expressed in the larval epidermal tissues from the day when the fifth instar larvae commenced with spinning behavior.
Protein Expression and Detection of the Chitinase Activity-The four cDNA constructs generated by alternative pre-mRNA splicing of the silkworm chitinase gene were expressed in E. coli strain Tuner(DE3)pLacI. After induction with IPTG, the total cell proteins from induced and uninduced transformed cell were subjected to SDS-PAGE. Intense bands of about 60 kDa were observed (Fig. 9A). Chitinase activity was assayed with glycol chitin-containing gel (32). The recombinant proteins produced active bands on the gel containing the substrate (Fig. 9B). DISCUSSION Sequence alignment of the coding regions in the B. mori genomic clone for the gene of chitinase (9) with the chitinase cDNA (7) revealed the correspondence of the two clones. However, the genomic clone incorporated an insert of 9 bp (5Ј-GTTCGTAAG-3Ј) at the C-terminal end of the gene, which was not reported in the chitinase cDNA (7). The fifth nucleotide of the 9 bp was found to be A instead of G in some of the cDNA clones, which may be due to sequencing ambiguities. It appears that these nucleotides increase the size of the encoded protein by three amino acids. In contrast, the protein encoded by this clone was found to be of smaller size when compared with the one deduced from the chitinase cDNA (7) because of the presence of a stop codon within the 9-bp insert DNA (Fig. 1).
Previous results of the amino acid sequence analysis of the silkworm chitinases (16) revealed that the first residue of the N-terminal sequence is the nonpolar aliphatic amino acid, al-FIG. 6. Multiple alignments of deduced amino acid sequences from B. mori chitinase cDNA clones obtained from different stages of the fifth instar larvae. Alignments were done using GENETYX-MAC software. Dots indicate identical amino acids, and dashes show the gaps introduced to preserve alignment. Identical amino acids are designated by the asterisk. The clones were obtained from the fifth instar larvae epidermal tissues 1, 2, 2.5, and 3 days after the spinning behavior and identified by SP-1, SP-2, SP-2.5, and SP-3, respectively. The amino acid sequences at the N-and C-terminal regions of the clones were aligned. Size differences within the clones were due to alternative splicing of the pre-mRNA.
anine. However, the reported cDNA clone (7) showed the first residue of the mature N-terminal sequence is the negatively charged amino acid, aspartate. Our results from the genomic clone of the B. mori chitinase gene suggested both cases to be true. To clarify the ambiguous results observed at the N-terminal region of the chitinase gene, to verify the existence of the 9-bp insert DNA in the mRNA, and to determine at which level these sequence differences are regulated, cDNA clones from the same insect species were synthesized at different stages of the fifth instar larvae and analyzed. Previously, we proposed that these discrepancies might be due to the insects' strains used in the different studies or otherwise regulated at the level of pre-mRNA splicing (9).
There are many genes reported to exhibit alternative RNA splicing in various organisms (34). Among insects, the tobacco hornworm, M. sexta, serpin gene produces 12 different serine proteinase inhibitors (serpins) through alternative pre-mRNA splicing (19,21). This splicing process generates inhibitor diversity and potentially regulates a variety of proteinases, using the same protein framework joined to different reactive site region cassettes (19). There are also about ten genes in Drosophila melanogaster found to exhibit different patterns of alternative splicing. The class I glutathione S-transferase (GST) gene family was also found to undergo alternative splicing in Anopheles gambiae (35) and Anopheles dirus (36). In both species the GST gene contains six exons for four mature GST transcripts, which share exons 1 and 2 but vary between four different exon 3 sequences (exons 3A-3D) (36). The sericin 1 primary transcript of B. mori is differentially spliced via a tissue-and developmentally regulated process (37). The structure of the sericin 1 gene is characterized by the presence of a large central alternative exon, which encodes an internally repetitive sequence (37). The serpin gene-1 from M. sexta is characterized by the presence of 12 alternate forms of exon 9. The splicing pathway apparently allows inclusion of only one exon 9 per molecule of a mature serpin-1 mRNA (21).
In this study we demonstrated that the B. mori chitinase gene exhibits four patterns of alternative pre-mRNA splicing: one at the 5Ј region and three at the 3Ј region (Figs. 5 and 6). In some of the cDNA clones amplified (SP-1/(1-10)2, SP-2/ (1-10)1, SP-2/(1-10)2, and SP-2/ (1-14)), the 3Ј border of exon 1 was found to be three nucleotides downstream (GTG/gtgagt), giving a more conventional intron entry site, gtgagt (37) ( Tables I and II and Fig. 1). In this case the gene undergoes differential splicing through two alternative 5Ј-intron splicing consensus sites at the boundary between exon 1 and intron 1 (Figs. 4 -6). Although the selection of the correct pairs of 5Ј-and 3Ј-intron splicing sites in pre-mRNA splicing process is the central problem, evidence is accumulating in support of the notion that combinations between certain intron entry sites are preferred to others (34). Based on this result, we were able to interpret that the discrepancies observed in the mature N-terminal sequences among the N-terminal amino acid sequence of B. mori chitinases (16), chitinase cDNAs (7,8), and the genomic clone for the gene of chitinase (9) were due to this alternative splicing process. The products of the splicing are two mature transcripts encoding two mature proteins: one starts with alanine and the other with aspartate. The splicing product that yields alanine as the first residue of the mature N-terminal increases the coding region of the chitinase gene by one amino acid, whereas the one that produces aspartate as the first residue of the mature N-terminal is one residue shorter. Exclusion or inclusion of the alternatively spliced triplet does not interrupt the ORF of the corresponding mature transcripts. It is noteworthy that the 5Ј region in the B. mori chitinase gene (9) is very unique compared with its closely related gene from M. sexta (18), both in sequence and structural motifs, because there is only one functional 5Ј-intron splicing consensus sequences in the junction between exon 1 and intron 1 in the M. sexta gene for chitinase (Table I). This might be attributed to species differences.
Another striking feature of the B. mori chitinase gene is its incorporation of 9 bp at the 3Ј region (9). This insertion mediates direct repeats of 112 bp. However, if the 9-bp insert DNA is summed up to the upstream 112 bp, the direct repeats will be 121 bp (Fig. 5). There are two types of splicing patterns in this region of the gene. A splicing occurs via exclusion of the upstream 121-bp direct repeat in some of the mature transcripts (SP-1/(1-10)2, SP-2/(1-10)2, and SP-2/(1-14)) (Table II), because the 3Ј-end of the 9-bp insert DNA serves as an alternative intron splicing site. The stop codon in the product of this splicing pattern was shifted to the downstream 121-bp direct repeat. The proteins encoded by either inclusion or exclusion of the upstream direct repeat will be of similar size in terms of their amino acids (Fig. 6).
To verify that the cDNAs described were produced by splicing and were not transcribed from additional gene sequences elsewhere in the genome, the same primer set was used in PCR with genomic DNA as the template to amplify the 3Ј region around the direct repeats. Nucleic acids that differ by 121 bp are detectable on agarose gel. The DNA fragments amplified from SP-0 through SP-3 genomic DNAs were typically the same size as expected (Fig. 7A). The same primer set was used in PCR with cDNA clones that exhibited differential splicing of the upstream 121-bp direct repeat as the templates (Fig. 7B). The results clearly indicated that the variant mature transcripts investigated are the products of a single gene, which in turn supports the concept that the copy number of B. mori chitinase gene is 1 per haploid genome (8). In addition, these results are inconsistent with the possibility that the nucleotides of the 9 bp are part of a large intron separating the repeated sequences. Moreover, the 9-bp insert DNA was also determined by sequencing to be alternatively spliced (Figs. 4 and 5). The mechanism by which the gene undergoes splicing of the 9 bp is not clear. However, the 9 bp may be spliced in a combinatorial fashion, because, in this pattern of splicing, the alternatively spliced genes contain entire exons that are individually included or excluded from the mature mRNA (34). The combinatorial exons, each 12-18 bp long, are among the smallest reported (34). However, the cardiac troponin T (TnT) gene has an exon of only six nucleotides in length (38). A previously reported B. mori chitinase cDNA (7) incorporates the 112-bp direct repeats but is devoid of the 9 bp, whereas the other reported cDNA from the same insect (8) lacks the upstream 121-bp direct repeat. All of these results together support the possibility of splicing of the 9 bp separately and irrespective of the other sequences of the primary transcript. Furthermore, exclusion of this insertion from the primary transcript yields a mature mRNA that encodes a protein 22 amino acids longer than that from a mature transcript including the insertion due to generation of a premature stop codon (Figs. 3 and 6). Exclusion of the triplet (GTG) at the exon 1 and intron 1 boundary and retention of the 9 bp at the 3Ј region appeared more commonly in the mature transcripts from the late stages of the fifth instar larvae. However, generally, the alternative splicing phenomenon in the B. mori chitinase gene is not dependent on the fifth instar developmental stage (Table II).
To assess the changes in the levels of chitinase mRNAs in the fifth instar larvae epidermal tissues, total RNAs from the re-  (1-8), respectively. The proteins were stained with Coomassie brilliant blue R-250. Molecular mass markers are shown in kilodaltons. B, native-PAGE and chitinase activity staining. Proteins from the inclusion bodies of the IPTG-induced or uninduced expression host cultures carrying the above cDNA constructs were solubilized and refolded as described under "Experimental Procedures." After native-PAGE, the proteins were transblotted onto a 7.5% polyacrylamide slab gel containing 0.01% glycol chitin as substrate and stained with Fluostain I. Lanes 1-4 contain induced chitinases in the expression host transformed with the cDNA clones SP-1/(1-10)1, SP-2/(1-10)2, SP-2/(1-10)1, and SP-2.5/ (1-8), respectively. Lane 5 contains induced cells of the expression host, Tuner, transformed with a control plasmid (Novagen). Lanes 6 -9 contain protein extracted from uninduced cultures of the expression host transformed with the cDNA clones SP-1/(1-10)1, SP-2/(1-10)2, SP-2/(1-10)1, and SP-2.5/(1-8), respectively. spective tissues were analyzed by Northern blotting. Chitinase mRNA was undetected by Northern blotting on the day when the fifth instar larvae started spinning behavior. However, it was present at a very low level 1 day later, and its level increased sharply on the second day and gradually onward (Fig. 8A). The mature mRNAs expressing chitinase were also detected by RT-PCR (Fig. 8B). These data demonstrated the chitinase mRNA was transcribed even at the SP-0 stage, and it was detectable by RT-PCR when using only half of the amount of total RNA that was used in Northern blotting. This result strongly supports the concept of a gradual expression of the chitinase mRNA in B. mori from the day when the fifth instar larvae initiated spinning behavior and also supports the development-specific expression of chitinase, which was also detected in M. sexta larvae (6).
Two mechanisms were proposed for the generation of multiple functional chitinases from a single gene in B. mori. The chitinases may be processed from a larger one by limited proteolysis from the C-terminal side (16). Another mechanism is the regulation of the enzymes expression at the gene level through alternative splicing of pre-mRNA. We present here the data that prove the alternative splicing mechanism. The role of this splicing mechanism in generating multiple functional chitinases in the silkworm was further demonstrated by expressing the alternatively spliced cDNA clones in E. coli. Chitinase activity of the four constructs was detected (Fig. 9B). This result reveals the role of alternative splicing of the primary transcript of the B. mori gene for chitinase in generation of multiple functional chitinase isoforms from a single gene, which is also consistent with three active chitinase isozymes expressed in vivo (16,17). The post-translational modifications of M. sexta chitinase (39) and the glycosylation of the molting fluid chitinase (40) also give more support to this finding.
In conclusion, this study reports the involvement of alternative splicing of the primary transcript in generating multiple functional chitinase isoforms in the silkworm. The presence of this splicing mechanism in the B. mori gene for the chitinase also contributes to the interpretation of the variations reported in studies of the insect's cDNAs. All of the mRNA products are from a single gene and functionally active.