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J. Biochem, 2004, Vol. 135, No. 2 231-236
© 2004 The Japanese Biochemical Society

BIOCHEMISTRY

Structure of a cDNA for Ciona Cytochrome b5 and the Ubiquitous Expression of mRNA in Embryonic Tissues

Toshitsugu Yubisui*,1, Takashige Takabayashi2, Fumi Takahashi2, Shigeki Fujiwara2 and Kazuo Kawamura2

1 Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005; and 2 Laboratory of Cellular and Molecular Biotechnology, Faculty of Science, Kochi University, Akebono-cho, Kochi 780-8520

A cDNA clone for cytochrome b5 was isolated from a cDNA library of an ascidian, Ciona savignyi, by a plaque hybridization method using a digoxigenin-labeled cDNA for the soluble form of human cytochrome b5. The cDNA is composed of 5'- and 3'-noncoding sequences, and a 396-base pair coding sequence. The 3'-noncoding sequence contains polyadenylation signal sequences. The amino acid sequence of 132 residues deduced from the nucleotide sequence of the cDNA showed 61% identity and 82% similarity to the cytochrome b5 of another ascidian species, Polyandrocarpa misakiensis, which we previously cloned. The amino-terminal hydrophilic domain of 98 residues contains well-conserved structures around two histidine residues for heme binding. A cDNA expression system was constructed to prepare a putative soluble form of Ciona cytochrome b5. The recombinant soluble cytochrome b5 showed an asymmetrical absorption spectrum at 560 nm as is shown by mammalian cytochromes b5 upon reduction with NADH and NADH-cytochrome b5 reductase. The recombinant Ciona cytochrome b5 is reduced by NADH-cytochrome b5 reductase with an apparent Km value of 3.3 µM. This value is similar to that of the cytochrome b5 of Polyandrocarpa misakiensis. The expression of Ciona cytochrome b5 mRNA during development was examined by an in situ hybridization method and ubiquitous expression in embryonic tissues was observed. The results indicate that cytochrome b5 plays important roles in various metabolic processes during development.

* To whom correspondence should be addressed. Tel/Fax: +81-86-256-9729, E-mail: tyubi{at}dbc.ous.ac.jp


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