| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1993: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
The Fasciola cDNA library was constructed in lambdagt11 and the screening of 4 x 10^4 independent plaques resulted in two positive clones. These clones (B22-2 and B7-2), which contained 1.1 kb cDNA inserts, were sequenced. Two cDNA clones encoded the Fasciola cysteine protease precursors. The deduced precursors contained 325 - 326 amino acid residues consisting of a signal sequence (pre-region, 15 residues), pro-region (90 residues), and mature protease (220 - 221 residues, Mr.24kDa). Cys^<27>, His^<164>, and Asn^<184> form the catalytic triad in the active side of the mature enzyme, and their adjacent regions were highly conserved. The parasite enzymes showed 50% amino acid sequence homologies, compared with mammalian lysosomal cathepsin L.Interestingly, asparadgine-linked glycosylation sites wich are thought to be targeting signals for cathepsins into lysosomes is absent in any region of the Fasciola cysteine protease (cathepsin L) molecule. The fact that two Fasciola Cathepsin L hav
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e no glycosylation motif is strongly suggestive of the occurrence of a different mechanism by which the Fasciola cathepsin L are transported into secretory granules of intestinal epithelial cells. Thus, to elucidate the reguratory mechanism of Fasciola cathepsin L gene expression and targeting to secretory granules. We characterized the gene structure of the Fasciola cathepsin L.
Two structural genes encoding Fasciola cathepsin L were amplified by polymerase chain reaction (PCR) using oligonucleotide primers synthesized on the basis of the 5'- and 3' of two cDNA open reading flames and Fasciola genomic DNA.The PCR-amplified products (1.9 and 1.8 kb) were sequenced. Both 1.9 kb-size and 1.8 kb-sizecathepsin L gene contained 4 exons and 3 introns. Exon 1 encoded the signal sequence, pro-region, and N-terminal region (21 amino acid residues) of the mature enzymes. Exons 2-4 encoded the remaining region of the mature enzyme, indicating that the structures of cathepsin L do not correspond to functional units as is the cases in other cathepsin genes. The lengths of the introns were 53-58,158-161, and 694-582bp, respectively. The third introns were quite different from the size and sequences. Interestingly, the Fasciola cathepsin L genes consist of a multigene family, although the mammalian cathepsin L gene is a single gene. Less
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