The Chitinase Gene of the Silkworm, Bombyx mori, Contains a Novel Tc-like Transposable Element

Abstract

We have determined the cDNA sequence and the genomic organization of the chitinase gene of the silkworm, Bombyx mori. The cDNA encodes 544 amino acids having 83% amino acid homology to the chitinase of the tobacoo hornworm, Manduca sexta. The total length of the gene is larger than 25 kilobase pairs, and it is separated into 11 exons. The intron-exon boundaries are all in accordance with the GT-AG rule. Also, the TATA box sequence was found in the 5 upstream region of the gene, and the gene is mapped on the seventh chromosome. A novel DNA type transposon that shows similarity to the Tc-like element was found in the third intron in some strains of B. mori; other strains, however, lack this element in the same intron. This element has long terminal inverted repeats, presumably encodes a transposase of about 340 amino acids with a DDE motif, and has an amino-terminal domain with a strong nuclear localization function. Seven other transposable elements with homologous but distinct sequences were isolated from the B. mori genome. Together with plaque hybridization results, our findings suggest that these novel elements exist in multiple copies constituting a new Tc-like transposable element family in the silkworm genome.Chitin is a ␤(1,4)-linked polymer of N-acetylglucosamine believed to be the second largest bio-mass next to cellulose. Insects utilize it as the major component of their exoskelton. When bound to proteins, chitin is strong enough both mechanically and biochemically to protect and light enough to allow smooth locomotion. However, once made as a component of the integument, chitin, unlike bone in vertebrates, has no growth capability, and the insects have to molt or undergo metamorphosis to reconstruct their integuments. Insect chitinase (family number 18 of glycosyl hydrolases, endochitinase) is induced by ecdysteroids at the time of molting and metamorphosis of the larvae to degrade most of the older chitin (1, 2). Further hydrolysis of the partially digested chitin is done by ␤1,4-Nacetylglucosaminidase (exochitinase) that is also inducible by ecdysteroid (3). The recycling of ␤1,4-N-acetylglucosamine from the older integument to the new integument was shown in larval-adult molting of Locusta migratoria (4) and larval-larval molting of Drosophila melanogaster (5). From insects, the chitinase cDNA of the tobacco hormworm Manduca sexta, has been isolated (6). With more than 4000 years of domestication history (7), Bombyx mori is one of the genetically most well studied organisms and is also a suitable model for hormone research. The existence of the molting hormone secreted from the prothoracic gland was experimentally shown using B. mori pupae (8, 9). The molting hormone, ecdysone, was isolated from the pupae of B. mori and crystallized, and its chemical structure was determined (10). The existence of the neuropeptide called prothoracicotropic hormone that stimulates the release of ecdysone was predicted (11), and the hormone was finally isolated and characterized (12).Here we describe the cDNA clonin...

Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases

“…In all insect chitinases sequenced so far, a hydrophobic signal peptide is predicted to precede the N-terminal region of the mature protein (Kramer et al, 1993;Koga et al, 1997;Choi et al, 1997; Nielsen et al, 1997; Shen and Jacobs-Lorena, 1997; Kim et al, 1998;Mikitani et al, 2000;Royer et al, 2002). The signal peptide presumably mediates secretion of the enzyme into the endoplasmic reticulum and it is cleaved off by signal peptidases after the protein has been transported across the membrane (von Heijne, 1990;Müller, 1992).…”

Alternative Splicing of the Primary Transcript Generates Heterogeneity within the Products of the Gene for Bombyx mori Chitinase

“…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 * This work was supported by a grant-in aid from the Ministry of Education, Science, Sports and Culture of Japan.…”

“…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).…”

“…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).…”

Low transposition frequency of the medaka fish Tol2 element may be due to extranuclear localization of its transposase

“…Because the elements, substrates for transposases, are present as parts of the chromosomes, transposase proteins need to be transported into the nucleus after they are synthesized on the ribosomes in the cytoplasm. Many proteins that act in the nucleus are known to contain NLSs (Nair et al, 2003), and this is also the case for some highly active DNAbased elements such as Activator of maize (Boehm et al, 1995), Mu of maize (Ono et al, 2002), Tag1 of Arabidopsis (Liu and Crawford, 1998), mariner of Drosophila and BmTc1 of the silkworm (Mikitani et al, 2000).…”