Molecular cloning of cDNA encoding APC11, a catalytic component of anaphase-promoting-complex (APC/C), from goldfish (Carassius auratus), and establishment of in vitro ubiquitinating system.
Destruction of cyclin B is required for exit from mitosis and meiosis. A cyclin-degrading system, including anaphase-promoting-complex/cyclosome (APC/C), has been shown to be responsible for cyclin B destruction. Here we present the cloning, sequencing, and expression analysis of goldfish (Carassius auratus) APC11, which encodes the catalytic component of APC/C from goldfish ovary. The cloned cDNA is 348 bp long and encodes 88 amino acids. The deduced amino acid sequence is highly homologous to APC11 from other species. The expression of mRNA for APC11 was ubiquitous among tissues, as opposed to that of mRNA for E2-C, which occurred at a very high level in the ovary. Recombinant goldfish APC11 possesses ubiquitinating activity against cyclin B. We established an in vitro ubiquitinating system of proteins composed of purified recombinant E1, E2-C, and APC11 from goldfish. The reconstructed system for these ubiquitinating enzymes makes it feasible to elucidate the molecular mechanism of cyclin B degradation.
Tokumoto
Mika
M
Department of Biology and Geosciences, Faculty of Science, National University Corporation Shizuoka University, Japan.
Kurita
Yoshihisa
Y
Tokumoto
Toshinobu
T
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D019926:Cyclin B / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006054:Goldfish / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010053:Ovary / Q000201:enzymology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D019926:Cyclin B; D018076:DNA, Complementary; D012333:RNA, Messenger; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome