In this study, to develop a technique that enables authentication of processed seafood, the complete nucleotide sequence of the mitochondrial genome for the Japanese flying fish Cypselurus hiraii was determined. Three segments spanning the entire genome were amplified using polymerase chain reaction, and products were subsequently used as templates for direct sequencing with 60 primers. The genome (16 528 base pairs) was found to contain the same 37 genes (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as those found in other vertebrate mitochondrial genomes, with the gene order being identical to that typical of vertebrates. A major noncoding region between the tRNA Pro and tRNA Phe genes (868 base pairs) appears to be the control (Dloop) region, as it has several conserved blocks characteristic of control regions.KEY WORDS: authentication of processed food, mitogenomics, mitochondrial DNA, polymerase chain reaction.

In the current studies, we sequenced and characterized the genomic and complementary deoxyribonucleic acid clones encoding the cellobiohydrolase encoding genes cel1 and cel2 of Polyporus arcularius. The predicted amino acid sequences of Cel1 and Cel2 are similar to glycosyl hydrolase family 7 and 6 proteins, respectively. The expression of cel1 and cel2 was induced by microcrystalline cellulose (Avicel) and cellopentaose but repressed by glucose, cellobiose, cellotriose, and cellotetraose. There was a very low level of cel1 and cel2 transcription regardless of the carbon source. These results suggest that P. arcularius cells constitutively express a very low level of cellulase that can degrade insoluble crystalline cellulose and that the transcription of cel1 and cel2 in the cells is induced by products produced by these endoglucanases such as cellooligosaccharides.

Nucleotide sequence and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the 3 0 -portion of the mitochondrial 16S RNA gene (rDNA) coding sequence were used to differentiate between flying fishes and other fishes that are frequently used in ago-noyaki production. In this study, we successfully distinguished between flying fishes and the other fishes by combining amplified DNA fragments with universally designed primers and digesting the PCR products with AfaI and MfeI restriction endonucleases. PCR-RFLP of 15 ago-noyaki, 2 noyaki, and 8 other processed flying-fish products were analyzed to authenticate flyingfish content among processed seafood products. After digestion of the PCR products with both enzymes, we found that all ago-noyaki and processed flying fish products contained either two or three DNA fragments of *200, 300, and 530 bp, and noyaki samples (which do not contain flying fish) contained only one fragment of *530 bp. Here, we present a new procedure to confirm the content of flying-fish meal in ago-noyaki.

Real-time polymerase chain reaction (PCR) analysis of the 3 0 -portion of the mitochondrial 16S RNA gene (rDNA) coding sequence was used to determine flying fish paste in ago-noyaki. We quantified the amount of flying fish paste in ago-noyaki samples using flying fishspecific primers (Tobi16SF3/Tobi16SR) and universal primers (Univ16SF2/Univ16SR2). Using real-time PCR of standard ago-noyaki, a standard equation was obtained (y = 1.08x -3.20; R 2 = 0.977). This equation was then used to estimate the relative flying fish paste contents of eight commercially available ago-noyaki and two similar products. These results verified that the ago-noyaki products that had already been labeled with the E-mark deserved this status.

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