Two zebrafish (Danio rerio) antizymes with different expression and activities

Abstract

Cellular polyamines are regulated by a unique feedback mechanism involving ornithine decarboxylase (ODC) antizyme. The synthesis of mammalian antizyme requires a programmed translational frameshift event induced by polyamines. Antizyme represses ODC, a key enzyme for polyamine synthesis, through accelerating enzyme degradation by the 26 S proteasome. Antizyme also inhibits the cellular uptake of polyamines. In the present study we isolated two distinct zebrafish (Danio rerio) antizyme cDNA clones (AZS and AZL) from an embryonic library. Their sequences revealed that both clones required translational frameshifting for expression. Taking account of +1 frameshifting, AZS and AZL products were 214 and 218 residues long respectively and shared 51.8% amino acid identity. In rabbit reticulocyte lysates, both mRNA species were translated through spermidine-induced frameshifting. The presence of the two antizyme mRNA species in embryos, adult fish and a cultured cell line was confirmed by Northern blot analysis. The ratio of AZS mRNA to AZL mRNA in the adult fish was 1.8-fold higher than in the embryos. Whole-mount hybridization in situ demonstrated that both mRNA species are expressed in every tissue in embryo, but predominantly in the central nervous system and the eyes. Bacterial expression products of both cDNA species inhibited ODC activity, but only the AZS product accelerated ODC degradation in vitro. These results show that both zebrafish antizymes are induced by polyamines but their mRNA species are expressed differently during development. The difference in activities on ODC degradation suggests their functional divergence.

Urea cycle enzymes through the development of pacu (Piaractus mesopotamicus): the role of ornithine carbamoyl transferase

“…The ornithine concentration can be sufficient to supply polyamine synthesis during the development, since the maintenance of the cell polyamine concentration for growing, proliferation (Pilz et al 1990) and DNA synthesis is dependent on it and is altered by the concentration of its precursor, ornithine (Byus and Wu 1991). The ODC gene is housekeeping, and the putrescine production is regulated by polyamine levels in the ODC translation (Matsufuji et al 1996, Saito et al 2000. The ornithine concentration to polyamine synthesis can be directly affected by OCT activity.…”

Nuclear translocation of antizyme and expression of ornithine decarboxylase and antizyme are developmentally regulated

“…A lot of attention has recently been focused on AZ, because of its unique regulation by translational frameshifting. Thus far, the only study on embryonal Az/AZ expression in vertebrates has been performed on 24-h zebrafish embryos (Saito et al, 2000). Using whole-mount in situ hybridization it was demonstrated that Az mRNA is expressed in every tissue of the zebrafish embryo, but predominantly in the central nervous system and in the eyes.…”

Development of two bone-derived cell lines from the marine teleost Sparus aurata ; evidence for extracellular matrix mineralization and cell-type-specific expression of matrix Gla protein and osteocalcin

“…VSa13 and VSa16 cultures were initiated at 22C in L-15, a medium well suited for supporting cell growth in non-CO 2 -equilibrated environments and widely used to culture fish cells (Bols et al 1994;Chi et al 1999;Fernandez et al 1993;Ganassin et al 1999;Saito et al 2000;Tong et al 1997). In this medium, both cell types exhibited a spindle-like phenotype and a slow growth rate and were unable to mineralize their ECM.…”