Characterization and regulation of Schizosaccharomyces pombe gene encoding thioredoxin

A cDNA coding thioredoxin (TRX) was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization. The 438 bp EcoRI fragment, which was detected by Southern hybridization, reveals an open reading frame which encodes a protein of 103 amino acids. The genomic DNA encoding TRX was also isolated from S. pombe chromosomal DNA using PCR. The cloned sequence contains 1795 bp and encodes a protein of 103 amino acids. However, the C-terminal region obtained from the cDNA clone is -Val-Arg-Leu-Asn-Arg-Ser-Leu, whereas the C-terminal region deduced from the genomic DNA appears to contain -Ala-Ser-Ile-Lys-Ala-Asn-Leu. This indicates that S. pombe cells contain two kinds of TRX genes which have dissimilar amino acid sequences only at the C-terminal regions. The heterologous TRX 1C produced from the cDNA clone could be used as a subunit of T7 DNA polymerase, while the TRX 1G from the genomic DNA could not. The upstream sequence and the region encoding the N-terminal 18 amino acids of the genomic DNA were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357 to generate the fusion plasmid pYKT24. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by hydrogen peroxide, menadione and aluminum chloride. It indicates that the expression of the cloned TRX gene is induced by oxidative stress.     

A novel DNA-binding motif in the nuclear matrix attachment DNA-binding protein SATB1.

The nuclear matrix attachment DNA (MAR) binding protein SATB1 is a sequence context-specific binding protein that binds in the minor groove, making virtually no contact with the DNA bases. The SATB1 binding sites consist of a special AT-rich sequence context in which one strand is well-mixed A's, T's, and C's, excluding G's (ATC sequences), which is typically found in clusters within different MARs. To determine the extent of conservation of the SATB1 gene among different species, we cloned a mouse homolog of the human STAB1 cDNA from a cDNA expression library of the mouse thymus, the tissue in which this protein is predominantly expressed. This mouse cDNA encodes a 764-amino-acid protein with a 98% homology in amino acid sequence to the human SATB1 originally cloned from testis. To characterize the DNA binding domain of this novel class of protein, we used the mouse SATB1 cDNA and delineated a 150-amino-acid polypeptide as the binding domain. This region confers full DNA binding activity, recognizes the specific sequence context, and makes direct contact with DNA at the same nucleotides as the whole protein. This DNA binding domain contains a novel DNA binding motif: when no more than 21 amino acids at either the N- or C-terminal end of the binding domain are deleted, the majority of the DNA binding activity is lost. The concomitant presence of both terminal sequences is mandatory for binding. These two terminal regions consist of hydrophilic amino acids and share homologous sequences that are different from those of any known DNA binding motifs. We propose that the DNA binding region of SATB1 extends its two terminal regions toward DNA to make direct contact with DNA.     

Discovery of new human beta-defensins using a genomics-based approach

Epithelial beta-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a <1 Mb region of chromosome 8p22-p23. To identify new defensin genes, the DNA sequence from a contig of large-insert genomic clones from the region containing human beta-defensin-2 (HBD-2) was analyzed for the presence of defensin genes. This sequence survey identified a novel beta-defensin, termed HBD-3. The HBD-3 gene contains two exons, is located 13 kb upstream from the HBD-2 gene, and it is transcribed in the same direction. A partial HBD-3 cDNA clone was amplified from cDNA derived from IL-1beta induced fetal lung tissue. The cDNA sequence encodes for a 67 amino acid peptide that is approximately 43% identical to HBD-2 and shares the beta-defensin six cysteine motif. By PCR analysis of two commercial cDNA panels, HBD-3 expression was detected in adult heart, skeletal muscle, placenta and in fetal thymus. From RT-PCR experiments, HBD-3 expression was observed in skin, esophagus, gingival keratinocytes, placenta and trachea. Furthermore, in fetal lung explants and gingival keratinocytes, HBD-3 mRNA expression was induced by IL-1beta. Additional sequence analysis identified the HE2 (human epididymis secretory protein) gene 17 kb upstream from the HBD-3 gene. One splice variant of this gene (HE2beta1) encodes a beta-defensin consensus cysteine motif, suggesting it represents a defensin gene product. HE2beta1 mRNA expression was detected in gingival keratinocytes and bronchial epithelia using RT-PCR analysis. The discovery of these novel beta-defensin genes may allow further understanding of the role of defensins in host immunity at mucosal surfaces.     

Molecular cloning and expression of rat interleukin-1 alpha cDNA

A cDNA sequence coding for rat interleukin-1 alpha (IL-1 alpha) has been isolated from a cDNA library that was prepared with mRNA derived from LPS-stimulated rat peritoneal macrophages by using human IL-1 alpha cDNA as a probe. The rat cDNA encodes a 270 amino acid residue protein which is homologous (65%) to human IL-1 alpha. The rat cDNA sequence under SV40 early promoter directed the synthesis of biologically active IL-1 in monkey COS-1 cells. Rat IL-1 alpha mRNA is not expressed in spleen, lung, liver or brain, and is also not expressed in these organs of LPS-treated rat except spleen. This suggests that IL-1 alpha is not produced constitutively in various tissues and LPS is not sufficient to induce IL-1 alpha in most tissues. Our data indicate that the IL-1 activities which have been reported to be produced in the brain are not of alpha type. We have constructed a plasmid expressing the carboxy terminal 156 amino acids in Escherichia coli. Recombinant rat IL-1 alpha produced in COS cells or E. coli has cytotoxic activity against the human melanoma cell line A375S1 (GIF activity), which has been reported to be sensitive to human IL-1 alpha and IL-1 beta. This suggests that GIF activity is common to IL-1s derived from various sources.     

A circularly permuted myoglobin possesses a folded structure and ligand binding similar to those of the wild-type protein but with a reduced thermodynamic stability

A circular permutein of sperm whale myoglobin in which the G helix is C-terminal, the H helix is N-terminal, and 16 amino acids link the H helix to the A helix has been expressed in Escherichia coli. The permutein sequence begins with Gly121 (using the numbering scheme for the wild-type protein) and terminates with Pro120. The ligand binding function of the permutein was assayed using stopped-flow methods and shown to be essentially identical to that of the wild-type protein. In addition, one- and two-dimensional NMR studies of the cyanomet isoform of the permutein show a nativelike structure with a heme binding pocket very similar to that of the wild-type myoglobin. Although the structure and function of the permutein resemble those of the wild-type myoglobin, the permutein is less stable to chemical denaturation by 5.2 kcal/mol.     

cDNA cloning, genomic structure, and chromosomal localization of a novel murine epidermis-type lipoxygenase.

Using a combination of degenerate PCR technique and conventional screening procedures, we isolated a cDNA encoding a novel lipoxygenase, termed epidermis-type lipoxygenase-3 (e-LOX-3, gene symbol Aloxe3), from mouse skin. Aloxe3 mRNA is expressed in the stratified epithelia of skin, tongue, and forestomach. The cDNA encodes a protein of 711 amino acids with a calculated molecular mass of 80.6 kDa. The amino acid sequence shows approximately 54% identity to the recently identified 12(R)-lipoxygenase. Sequence comparison revealed a segment of 41 amino acid residues localized near the boundary between the N- and the C-terminal domain sequences of the molecule, a structural feature that is also characteristic of 12(R)-lipoxygenase, suggesting that these two epidermis-derived lipoxygenases may be members of a novel structural class of mammalian lipoxygenases. The novel lipoxygenase gene is divided into 15 exons and 14 introns, spanning 22.3 kb of genomic DNA. By interspecific backcross analysis, the novel gene was localized to the central region of mouse chromosome 11.     

An Arabidopsis cDNA encoding a DNA-binding protein that is highly similar to the DEAH family of RNA/DNA helicase genes.

A cDNA encoding a putative RNA and/or DNA helicase has been isolated from Arabidopsis thaliana cDNA libraries. The cloned cDNA is 5166 bases long, and its largest open reading frame encodes 1538 amino acids. The central region of the predicted protein is homologous to a group of nucleic acid helicases from the DEAD/H family. However, the N- and C-terminal regions of the Arabidopsis cDNA product are distinct from these animal DEIH proteins. We have found that the C-terminal region contains three characteristic sequences: (i) two DNA-binding segments that form a probe helix (PH) involved in DNA recognition; (ii) an SV40-type nuclear localization signal; and (iii) 11 novel tandem-repeat sequences each consisting of about 28 amino acids. We have designated this cDNA as NIH (nuclear DEIH-boxhelicase). Functional character-ization of a recombinant fusion product containing the repeated region indicates that NIH may form homodimers, and that this is the active form in solution. Based on this information and the observation that the sequence homology is limited to the DEAH regions, we conclude that the biological roles of the plant helicase NIH differ from those of the animal DEIH family.     

Isolation and characterization of a cDNA encoding a synaptonemal complex protein.

A gene encoding a 65-kilodalton antigen of the rat synaptonemal complex, SC65, has been cloned by screening rat testis lambda gt11 and lambda ZAPII cDNA expression libraries using polyclonal antibodies against rat synaptonemal complex proteins. The longest open reading frame, initiating at an ATG codon in the cDNA, encodes a protein of 431 amino acids, with a relative molecular mass of 50,000. Immunological analysis locates the SC65 gene product on the synaptonemal complex between the pairing faces of the parallel aligned cores of homologous chromosomes in spermatocytes. Of the rat tissues examined, the SC65 gene is transcribed in testis, brain, and heart at similar levels, and in the liver at a much lower level. The DNA sequence extending about 80 base pairs downstream of the translation termination codon has 93% similarity to the identifier sequence present in the rat genome in 1 x 10(5)-1.5 x 10(5) copies and in cDNA clones of precursors of brain-specific mRNAs. The amino acid sequence encoded by the SC65 gene contains an acidic region in the C-terminal domain of the protein, potential glycosylation sites, and at least one possible phosphorylation site. The protein shows no overall similarity to proteins of known function, nor is there similarity to protein sequences present in GenBank or EMBL data bases.     

Molecular cloning and expression in Escherichia coli of a cDNA encoding human pancreatic elastase 2

We have cloned a DNA that is complementary to the messenger RNA that encodes human pancreatic elastase 2 from a human pancreatic cDNA library using a cloned cDNA for rat pancreatic elastase 2 messenger RNA. This complementary DNA contains the entire protein coding region of 807 nucleotides which encodes preproelastase of 269 amino acids, and 4 and 82 nucleotides of the 5'- and 3'-untranslated sequences, respectively. When this deduced amino acid sequence was compared with known amino acid sequences it showed 82% homology with rat pancreatic elastase 2. This deduced sequence also contains a 16-amino-acid peptide identical with the N-terminal sequence determined for native human pancreatic proelastase 2. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 241 amino acids including 16 and 12 amino acids for a signal peptide and an activation peptide, respectively. Moreover, the predicted key amino acid residues involved in determining the substrate specificity of mammalian pancreatic elastase 2 are retained in the human enzyme. Cloned human pancreatic elastase 2 cDNA was expressed in E. coli as a mature and pro-form protein. Both resulting proteins showed immunoreactivity toward anti-elastase serum and enzymatic activity. We have also cloned and sequenced a porcine pancreatic elastase 2 cDNA.     

Molecular cloning and expression in Saccharomyces cerevisiae of a laccase gene from the ascomycete Melanocarpus albomyces

The lac1 gene encoding an extracellular laccase was isolated from the thermophilic fungus Melanocarpus albomyces. This gene has five introns, and it encodes a protein consisting of 623 amino acids. The deduced amino acid sequence of the laccase was shown to have high homology with laccases from other ascomycetes. In addition to removal of a putative 22-amino-acid signal sequence and a 28-residue propeptide, maturation of the translation product of lac1 was shown to involve cleavage of a C-terminal 14-amino-acid extension. M. albomyces lac1 cDNA was expressed in Saccharomyces cerevisiae under the inducible GAL1 promoter. Extremely low production was obtained with the expression construct containing laccase cDNA with its own signal and propeptide sequences. The activity levels were significantly improved by replacing these sequences with the prepro sequence of the S. cerevisiae alpha-factor gene. The role of the C-terminal extension in laccase production in S. cerevisiae was also studied. Laccase production was increased sixfold with the modified cDNA that had a stop codon after the native processing site at the C terminus.     

Isolation and characterization of a complementary DNA clone coding for the E1 beta subunit of the bovine branched-chain alpha-ketoacid dehydrogenase complex: complete amino acid sequence of the precursor protein and its proteolytic processing

A 1.7-kb cDNA clone encoding the entire precursor of the E1 beta subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex was isolated from a bovine liver cDNA library by screening with a mixture of synthetic oligonucleotide probes corresponding to the C-terminal five-residue sequence of the mature E1 beta subunit. A partial amino acid sequence was determined by Edman degradation of the intact subunit and the peptides generated by cleavage at the lysyl bonds. Nucleotide sequence analysis revealed that the isolated cDNA clone contained the 5'-untranslated sequence of 186 nucleotides, the translated sequence of 1176 nucleotides, and the 3'-untranslated sequence of 306 nucleotides with a poly(A) tail. A type AATAAA polyadenylation signal was located 17 nucleotides upstream of the start of a poly(A) tail. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the partial amino acid sequence of the mature BCKDH E1 beta subunit showed that the cDNA insert encodes for a 342 amino acid subunit with Mr 37,745 and that the subunit is synthesized as the precursor with a leader sequence of 50 amino acids and processed at the N-terminus. Northern blot analysis using the cDNA insert as a probe showed the presence of a 1.8-1.9-kb mRNA in bovine liver, suggesting that the insert covers nearly a full length of mRNA. Alignment of the deduced amino acid sequence of bovine BCKDH E1 beta with that of the human pyruvate dehydrogenase (PDH) complex E1 beta subunit revealed a high degree of sequence homology throughout the two enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression in Escherichia coli of fully active recombinant human IL 1 beta: comparison with native human IL 1 beta

Although complementary DNA (cDNA) encoding human interleukin 1 beta (IL 1 beta) have been cloned in several laboratories, there are as yet no data demonstrating that recombinant IL 1 beta (rIL 1 beta) molecules expressed from such cDNA are faithful, fully active replicas of the native protein secreted by human monocytes. To this purpose, cDNA sequences corresponding to the exact NH2-terminus and amino acid sequence of mature, monocyte-derived human IL 1 beta were placed under control of the inducible trp-lac (TAC) fusion promoter and were expressed in E. coli strain JM105. rIL 1 beta was purified to homogeneity by high pressure liquid chromatography (HPLC). Yields of 10 to 20 mg of rIL 1 beta/liter/10(11) cells were obtained. Purified rIL 1 beta was then compared in a number of biochemical and biologic assays to purified native IL 1 beta. Native and rIL 1 beta co-migrated on SDS-polyacrylamide gels as 17.5 kd polypeptides and reacted with specific polyclonal antisera raised to three synthetic peptides of human IL 1 beta in immunoblot experiments. Amino acid sequence analysis of rIL 1 beta showed that the native amino terminus, an ALA residue, was faithfully maintained. Purified native and rIL 1 beta co-chromatographed on reverse-phase HPLC. Specific biologic activities of rIL 1 beta were indistinguishable from those of the native protein in murine thymocyte and human dermal fibroblast proliferation assays, with half-maximal stimulation occurring at concentrations of 25 pM in the murine thymocyte assay and 2 pM in the human dermal fibroblast assay. Similarly, native and rIL 1 beta competed equally well for high affinity IL 1 receptor binding sites, each exhibiting a Ki of 20 pM on MRC-5 human embryonic lung fibroblasts. These observations indicate that E. coli efficiently expresses large quantities of rIL 1 beta, which emulates exactly the properties of the native protein.     

Location of the cell-binding domain of CP65, a 65kDa cysteine proteinase involved in Trichomonas vaginalis cytotoxicity

The cysteine proteinase (CP) of 65 kDa, CP65, binds to the surface of HeLa cells and is involved in Trichomonas vaginalis cellular damage. To identify and locate the CP65 cellular-binding domain, we enriched the CP65 protein band by ammonium sulfate fractionation and ion-exchange chromatography and the N-terminal sequence was obtained. A 618 bp gene fragment was obtained by PCR using genomic DNA as template and primers derived from the N-terminal sequence of CP65 and the Asn papain-catalytic conserved region. This gene fragment encodes for 206 amino acid (aa) residues corresponding to the N-terminal region of a mature CP with 67–76% identity to the reported trichomonad cathepsin-L-like CPs. This gene fragment was expressed in a bacterial system for antibody production and functional analysis. Antibodies against the native trichomonad CP65 recognized the recombinant protein, referred to as rCP65, confirming its relationship with the CP65 gene. The rCP65 protein was bound to the surface of HeLa cells and competed with the native CP65 for binding. Antibodies to the rCP65 (-rCP65) reacted with the trichomonad CP65 located on the parasite surface, and inhibited trichomonal cytotoxicity in a concentration-dependent manner. These data strongly suggest that this gene fragment encodes for the putative cell-binding domain (CBD) of CP65 located at its N-terminal region.     

Molecular cDNA cloning and analysis of the organization and expression of the IL-1beta gene in the Nile tilapia, Oreochromis niloticus

The full-length cDNA sequence of interleukin-1beta (IL-1beta) from the Nile tilapia, Oreochromis niloticus, was determined by using PCR with primers designed from known fish IL-1beta sequences followed by elongation of the 5' and 3' ends using Rapid Amplification of cDNA Ends (RACE). The cDNA contains a 92-bp 5' untranslated region (UTR), a single open reading frame (ORF) of 732 bp that translates into a 243-amino acid molecule, a 341-bp 3' UTR with four cytokine RNA instability motifs (ATTTA), and a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly(A) tail. The organization of the genomic IL-1beta based on the cDNA sequence appeared to be 4 introns and 5 exons. In comparison with known IL-1beta amino acid sequences, including human, catshark, trout, turbot, carp, sea bream, sea bass and goldfish, the amino acid sequence deduced from the cDNA sequence of Nile tilapia showed different levels of identity ranging from 25.32% to 66.80% and homology ranging from 41.88% to 82.19%. Although the entire cDNA sequence of Nile tilapia IL-1beta showed from 49.45% to 67.05% identity to those of other reported IL-1beta cDNAs, each exon also showed different levels of identity to the corresponding exons of other reported IL-1beta cDNAs. The highest nucleotide sequence identity for exon 1 and exons 2-5 of Nile tilapia IL-1beta was found in the corresponding exons of sea bream and sea bass, respectively. After in vitro stimulation with lipopolysaccharide (LPS), we found an increased level of IL-1beta expression in head kidney cells compared to that of unstimulated cells. However, this difference was no longer apparent after 4 h of stimulation, at which time the levels were similar in stimulated and unstimulated cells. Head kidney cells stimulated in vivo by an intraperitoneal injection of LPS showed a peak level of IL-1beta expression after 1 day and a decreased level after 3 days. At 7 days after stimulation, we were hardly able to detect IL-1beta expression.     

Cloning and characterization of the cDNAs for human and rabbit interleukin-1 precursor.

DNA sequence complementary to the mRNA for rabbit interleukin-1 precursor (preIL-1) has been cloned from the cDNA library constructed using partially purified poly(A)+RNA from induced rabbit alveolar macrophages by mRNA hybridization-translation assay. By using this cDNA as a probe, human IL-1 cDNA was isolated from the cDNA library prepared using poly(A)+RNA from induced HL-60 cells, a human monocyte-like cell line. The amino acid sequences of the human and rabbit preIL-1 deduced from the cDNA sequences reveal their primary structures which consists of 271 and 267 amino acid residues, respectively. The amino acid sequence is 64% conserved between human and rabbit. The difference in number of amino acid residues results from the carboxy-terminal extention of 4 amino acid residues in human preIL-1. Expression of the cloned human cDNA in E. coli yielded biologically active IL-1.     

Characterization of an apoptosis inhibitory domain at the C-termini of FE65-like protein

TR2(L) is a 56-amino-acid polypeptide that has been shown to block TNF cytotoxicity. FE65-like (FE65L) proteins possess this conserved TR2(L) sequence at their C-termini, whereas variations in the sequences are found in the FE65 proteins. To further analyze the antiapoptotic function of TR2(L), here we utilized an isolated murine partial FE65L cDNA that encodes an N-terminal phosphotyrosine-binding domain (PTB) and the conserved C-terminal TR2(L) sequence. When L929 cells were stably transfected with the FE65L cDNA or its 3' end TR2(L) DNA sequence, these cells became resistant to TNF killing. Replacement of the N-terminal PTB domain with GFP failed to abolish the FE65L-mediated TNF resistance. Ablation of the C-terminal TR2(L) sequence through frame-shift mutation resulted in a complete loss of the FE65L function against TNF. Various protein kinase inhibitors, including lavendustin A, tyrphostin, H7, and staurosporine, which may affect the PTB domain function, could not abolish the FE65L-mediated TNF resistance. A prolonged exposure of L929 cells to these inhibitors for 24 h resulted in cell death, whereas FE65L significantly blocked the cell death. Polyclonal antibodies were generated against a synthetic peptide and shown to interact with a 38-kDa FE65L in L929 cells. Hyaluronidase downregulates the expression of FE65L gene and protein in L929 cells, and this correlates with its enhancement of TNF killing of these cells. Together, our data indicate that the TR2(L) amino acid sequence is an apoptosis-inhibitory domain commonly present in the FE65 and FE65-like family proteins.     

百合查尔酮合成酶基因的克隆与分析

以西伯利亚百合为试材,通过半巢式PCR和RT-PCR技术分别克隆了查尔酮合成酶基因(CHS)的DNA和cDNA.生物信息学分析显示,CHS的DNA序列全长1 397 bp(登录号HM622754),包含2个外显子和1个内含子;cDNA序列编码区全长1 182 bp(登录号HQ161731),编码393个氨基酸,具有3个典型的CHS蛋白结构域:N-末端结构域(Lys3-Pro229)、C-末端结构域(Gln239-Pro389)和聚合酶Ⅲ结构域(Met1-Thr391);不同百合品种的CHS基因编码的氨基酸序列相似性高达98%,表明百合CHS基因在进化上呈现出十分保守的趋势;不同植物CHS基因序列的系统进化邻接树结果表明:百合与单子叶植物鸢尾及禾本科的水稻、大麦、玉米等亲缘关系更为接近.     

Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli.

A cDNA clone coding for mature C. reinhardtii ferredoxin has been isolated from a cDNA library using PCR and two oligonucleotide primers based on the N- and C-termini of the protein's amino acid sequence. The nucleotidic sequence of the PCR fragment (299 bp) agreed well with the amino acid sequence since a single conservative substitution (Thr-7 to Ser) could be deduced. The PCR fragment was inserted into the expression vector pTrc 99A, using the incorporated NcoI and BamHI restriction sites and the construction used to transform E. coli (DH5 F′). After subsequent large scale expression and purification of the recombinant protein, biochemical and biophysical analysis have indicated that the product isolated from E. coli is homologous to native ferredoxin isolated from green algae.     

Cloning, sequencing, and expression of the tulip bulb chitinase-1 cDNA

A cDNA encoding tulip bulb chitinase-1 (TBC-1) was cloned using a combination of immunoscreening from a lambda ZAP cDNA library with anti-TBC-1 antiserum and the 5' rapid amplification of cDNA end (RACE) method, and sequenced. The cDNA consists of 1,106 nucleotides and included an open reading frame encoding a polypeptide of 314 amino acids. Comparison of the deduced amino acid sequence and the determined protein sequence indicated the presence of a signal peptide and an extra peptide composed of 26 and 13 amino acids at the N- and C-termini, respectively. The deduced sequence of TBC-1 had 10-20% and 63% sequence similarities to plant class III chitinases and gladiolus bulb class IIIb chitinase (GBC-a), respectively. The cDNA encoding mature TBC-1 was amplified by polymerase chain reaction (PCR), ligated into the expression vector pET-22b, and expressed in Escherichia coli BL21(DE3). The recombinant TBC-1 (rTBC-1) expressed in E. coli was purified by gel filtration followed by ion-exchange chromatography. Specific activity of the rTBC-1 was almost same as the authentic TBC-1 toward glycolchitin. This is the first report on the cDNA cloning of a class III chitinase having C-terminal extra peptide.