Isolation of total RNA and RT-PCR

Total RNA was isolated from a mixture of the tail fan and blade, and subjected to RT-PCR as described in Watanabe et al. (1996). Degenerate oligonucleotide primers CHIF (5′-ATGGTNKCNGT-NGGNGGNTGG-3′) and CHIR (5′-NGGRTAYTCCCARTCNANRTC-3′) were designed based on the amino acid sequences of the two conserved domains of the chitinases (Watanabe et al., 1996).

Construction and screening of a cDNA library from the tail fan/blade

Poly(A)⁺ RNA was prepared using Oligotex™-dT30 super (Roche Japan). A cDNA library was constructed from the poly(A)⁺ RNA as described in Watanabe et al. (1996) except that λZAPII (stratagene) was used instead of λgt11 as the cloning vector. A product of the RT-PCR reaction was cloned into the pCR™II vector (Invitrogen). The EcoRI fragment of this plasmid was self-ligated using T4 DNA ligase to generate longer fragments, radio-labeled with Multiprime™ DNA labeling system (Amersham), and used as a probe to screen the cDNA library. Conditions for hybridization and subsequent washing were described in Watanabe et al. (1996).

Nucleotide sequence analysis

Recombinant pBluescript SK⁻ phagemids were rescued from positive λ phage clones by in vivo excision according to the instruction from the manufacturer (stratagene). Insert DNA (3,357 bp) was sequenced as described in Watanabe et al. (1996). The nucleotide sequence of 1,229 bp in the 5′ untranslated region was determined only on the sense strand, and 2,128 bp on the 3′ side (containing the entire open reading frame; see Fig. 2) was sequenced on both strands. Homology search in the PIR and SWISS-PROT databases was carried out using the FASTA E-mailing service by DNA Data Bank of Japan (National Institute of Genetics, Mishima, Japan).

Northern hybridization

Poly(A)⁺ RNA (1 μg per lane) from a mixture of tail fan and blade, and hepatopancreas was run on 1% formaldehyde-agarose gel, transferred to NYTRAN membrane (Schleicher & Schuell), and probed with a cDNA fragment (corresponding to nucleotide position 1,362–2,972 in Fig. 2) that was radio-labeled with ‘α-³²P’dATP. The concentration of the probe in hybridization buffer was 5 × 10⁶ cpm/ml. The final wash of the membrane was done in 0.1 × SSPE and 0.1% SDS at 65°C. RNA ladder (0.24–9.5 kb; GIBCO BRL) was used as molecular weight markers.

Preparation of RNA from the tail fan and blade at late premolt stages and RT-PCR

Prawns at late premolt stages (D3 and D4) were identified according to Hong (1977), and total RNA was prepared from a mixture of the tail fan and blade which consist mainly of chitinous exoskeleton and underlying epidermis.

To isolate a cDNA fragment containing a coding sequence of a chitinase, the RNA sample was subjected to RT-PCR using two degenerate oligonucleotide primers CHIF and CHIR which were designed based on the amino acid sequences of the two conserved regions of the chitinase protein family (Watanabe et al., 1996). A RT-PCR product was cloned and sequenced (Fig. 1). A 111 base pair (bp) region which was bounded by the primer sequences was conceptually translated to 37 amino acids, and the sequence was compared to those of the M. sexta chitinase (Kramer et al., 1993) and Pjchi-1, a chitinase homologue in P. japonicus (Watanabe et al., 1996). In the 37 amino acid region, identity was found at 14 and 13 residues, respectively (data not shown), suggesting that the RT-PCR product contains a coding sequence of a chitinase homologue.

Fig. 1

Isolation and sequence analysis of a RT-PCR product. Total RNA from a mixture of the tail fan and blade was subjected to RT-PCR using two degenerate oligonucleotide primers CHIF and CHIR, and PCR products were cloned in pCR™II. Nucleotide sequence of a clone is shown here. Sequences corresponding to the primers are underlined, and the inferred amino acid sequence is shown below corresponding codons.

Fig. 2

Nucleotide sequence of the Pjchi-2 cDNA. Nucleotide sequence of a cDNA clone of 3,357 bp and conceptual translation of an open reading frame of 1,581 nucleotides (nucleotide position 1320–2960) are presented. The amino acid sequence is shown in the one-letter representation below the respective codons. The positions of the nucleotides and amino acids are indicated on the left of the sequences. A typical polyadenylation signal (AATAAA) was found 22 bases upstream from the poly(A) tail (underlined). An asterisk shows the termination codon.

Isolation and sequence analysis of a cDNA clone

This product was used as a probe to screen a cDNA library of the tail fan/blade at the late premolt stage. Six positive clones were isolated, and a clone containing the longest insert was selected for sequence analysis. Within this cDNA clone, an open reading frame (ORF) of 1,581 bp was found (Fig. 2) The ORF was conceptually translated to 527 amino acids, and this putative protein was named Pjchi-2 (Penaeus japonicus chitinase homologue-2) due to its sequence similarity to known chitinases (see below).

The first methionine in the ORF (amino acid position 1 in Fig. 2) is shortly preceded by a stop codon in all three reading frames (data not shown). The second and third methionines are found at amino acid positions 8 and 339, respectively (Fig. 2). Assuming that the first methionine is the translation start site, the predicted molecular weight of Pjchi-2 is 59.2 kDa and isoelectric point 8.19. A potential N-glycosylation site was found at amino acid position 230. We failed to find a typical signal peptide (von Heijne, 1986) at the N-terminus of Pjchi-2, as no noticeable stretch of hydrophobic amino acids was found following the first or second methionine.

Using the FASTA program (Pearson and Lipman, 1988), we searched in the PIR and SWISS-PROT databases for proteins which exhibit sequence similarities to Pjchi-2. Proteins with the five highest similarity scores were: (1) a chitinase precursor in a braconid wasp (optimized score 838; Krishnan et al., 1994), (2) a chitinase precursor in the tobacco hornworm Manduca sexta (775; Kramer et al., 1993), (3) the human cartilage glycoprotein gp39 precursor (762; Hakala et al., 1993), (4) a swine heparin-binding protein (757; Shackelton et al., 1995), and (5) a bovine oviduct-specific glycoprotein precursor (728; Sendai et al., 1994). The similarity score between Pjchi-2 and Pjchi-1 (Watanabe et al., 1996) was 695. In the optimized alignment between Pjchi-2 and each of the above six structurally related proteins, conserved amino acids were distributed more or less uniformly over a region of about 400 amino acids, and the identity level in the overlap was 35-40% (data not shown). Sequence similarity among Pjchi-2, Pjchi-1 and the two insect chitinases was particularly high in conserved region II (Fig. 3), the putative catalytic domain of the chitinases (Watanabe et al., 1993). Reasonable sequence similarity among those proteins was also seen in conserved region I (Fig. 3).

Fig. 3

Alignment of the Pjchi-2 sequence with two insect chitinases and Pjchi-1. The Pjchi-2 sequence in the vicinity of the two conserved regions of the chitinase family (Leu²¹⁵-Cys²⁷⁵) was aligned using the CLUSTAL V program (Higgins and Sharp, 1989) with a chitinase in a braconid wasp Chelonus sp. (Lys⁹⁰-Gln¹⁵⁹; Krishnan et al., 1994), a chitinase in the tobacco hornworm M. sexta (Ser⁹¹-Ala¹⁵⁰; Kramer et al., 1993), and Pjchi-1, a chitinase homologue in P. japonicus (Glu¹¹⁰-Ala¹⁶⁹; Watanabe et al., 1996). Positions of the two conserved regions are indicated above the sequences. Identity of amino acids between Pjchi-2 and the other proteins is indicated by dots, and a gap by a hyphen. The amino acids which are conserved in all of the proteins are indicated with asterisks.

Expression of the Pjchi-2 mRNA

Expression of the Pjchi-2 mRNA was examined, using Northern analysis, in the tail fan/blade and hepatopancreas (Fig. 4). Significant accumulation of transcripts was detected in the tail fan/blade during the premolt stage, whereas the expression level was much lower during the intermolt stage. The top of a smeary band was observed at approximately 3.2 kb (arrow in Fig. 4). The size of this band was in fair agreement with that of the cDNA clone (3,356 bp). The Pjchi-2 mRNA was not detected in the hepatopancreas.

Fig. 4

Northern analysis of Pjchi-2-encoding mRNA in cuticular tissues and the hepatopancreas. A Northern blot of poly(A)⁺ RNA (1 μg per lane) from a mixture of tail fan and blade (lanes 1 and 2), and the hepatopancreas (lanes 3 and 4) was hybridized with a radio-labeled cDNA probe which corresponds to nucleotide position 1,362–2,972 in Fig. 2. Samples in lanes 1 and 3 are from the intermolt stage (stage C according to Hong, 1977), and lanes 2 and 4 from late premolt stages (stages D3 and D4). A band was recognized at approximately 3.2 kb in lane 2 (arrow); this band was also seen in lane 1 after a long exposure. Positions of four molecular weight markers (7.5, 4.4, 2.4, and 1.4 kb, from top to bottom) are indicated.