Ascorbate oxidase expression in pumpkin (Cucurbita spp.) tissues was studied. Specific ascorbate oxidase activities in pumpkin leaf and stem tissues were about 2 and 1.5 times that in the fruit tissues, respectively. In seeds, little ascorbate oxidase activity was detected. Northern blot analyses showed an abundant ascorbate oxidase mRNA in leaf and stem tissues. Fruit tissues had lower levels of ascorbate oxidase mRNA than leaf and stem tissues. Ascorbate oxidase mRNA was not detected in seeds. Specific ascorbate oxidase activity gradually increased during early seedling growth of pumpkin seeds. The increase was accompanied by an increase in ascorbate oxidase mRNA. When ascorbate oxidase activity in developing pumpkin fruits was investigated, the activities in immature fruits that are rapidly growing at 0, 2, 4, and 7 d after anthesis were much higher than those in mature fruits at 14 and 30 d after anthesis. The specific activity and mRNA of ascorbate oxidase markedly increased after inoculation of pumpkin fruit tissues into Murashige and Skoog's culture medium in the presence of an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D) but not in the absence of 2,4-D. In the presence of 10 mg/L of 2,4-D, ascorbate oxidase mRNA was the most abundant. Thus, ascorbate oxidase is induced by 2,4-D. These results indicate that ascorbate oxidase is involved in cell growth. In pumpkin callus, ascorbate oxidase activity could be markedly increased by adding copper. Furthermore, immunological blotting showed that the amount of ascorbate oxidase protein was also increased by adding copper. However, northern blot analyses showed that ascorbate oxidase mRNA was not increased by adding copper. We suggest that copper may control ascorbate oxidase expression at translation or at a site after translation.Ascorbate oxidase (EC 1.10.3.3) is a copper-containing blue enzyme that has been studied in plant tissues such as pumpkin (17, 29), cucumber (24), and orange (31). The enzyme catalizes the oxidation of ascorbic acid to dehydroascorbic acid. Recently, the enzyme has been used for clinical and food analyses of L-ascorbic acid (8,20).The definitive biological function of ascorbate oxidase is not clear, although it has been reported that the enzyme may participate in a redox system involving ascorbic acids (32). Furthermore, the subcellular localization of ascorbate oxidase is uncertain. The enzyme may be localized in the cell wall 1 This work was supported in part by Grants-in-Aid for Scientific Research (No. 02261214) from the Ministry of Education, Science, and Culture of Japan. (13,22,25), although there have been some reports that the enzyme is localized in the cytoplasm (33), microsomes (33), or vacuole (31). Recently, Lin and Varner (18) studied the expression of ascorbate oxidase in zucchini and reported that the enzyme may be involved in reorganization of the cell wall.We have studied the ascorbate oxidase in cultured pumpkin (Cucurbita spp.) cells and in a previous paper reported that ascorbate oxidase activity ra...

A genomic clone encoding ascorbate oxidase was isolated from pumpkin (Cucurbita sp.)-This gene is consisted of four exons and three introns. Analyses of the promoter fusion to /J-glucuronidase reporter gene by transient expression assay in pumpkin fruit tissues suggested the existence of a cis-acting region responsible for auxin regulation.

Mutations in the JAG1 gene and the NOTCH2 gene cause Alagille syndrome. At present, however, genetic testing of Alagille syndrome is not commonly applied in clinical settings because the currently available assays are technically and financially demanding, mainly because of the size of the genes. In the present study, we optimized the highly sensitive and specific mutation scanning method automated denaturing high-performance liquid chromatography (DHPLC) to analyze the entire coding region of JAG1 and NOTCH2. The coding region was amplified by 69 primer pairs, all of which have the same cycling conditions, aliquoted on a 96-well format PCR plate. In this manner, all the exons were simultaneously amplified using a single block in a thermal cycler. We then wrote a computer script to analyze each segment of JAG1 and NOTCH2 by DHPLC in a serial manner using conditions that were optimized for each amplicon. The implementation of this screening method for JAG1 and NOTCH2 will help medical geneticists confirm their clinical impressions and provide accurate genetic counseling to the patients with Alagille syndrome and their families.

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