Cloning of ORF2 of antizyme, characterization, and relationship to regulation of ornithine decarboxylase during embryogenesis of Musca domestica

The second ORF of M. domestica antizyme was cloned and characterized. The second open-reading frame and the 3'-untranslated region contain 976 nucleotides that can be translated into a polypeptide of 198 amino acids. As the second ORF of antizyme has no initiation codon of its own, this represents only the 3'-end of the gene.;Regulation of antizyme mRNA throughout embryogenesis was evaluated using real-time PCR. Antizyme was found to be expressed above 200 fg/mug total RNA during early embryogenesis, followed by a slight reduction to approximately 200 fg/mug total RNA during mid-embryogenesis, and at levels generally below 200 fg/mug total RNA throughout the end of development.;Evaluation of ODC mRNA was performed using real-time PCR. Analysis of the ODC gene revealed that ODC levels are nearly undetectable until mid-embryogenesis, followed by a dramatic increase, with hour 7.0 postoviposition representing the peak of ODC mRNA expression. During the second half of embryogenesis, ODC mRNA levels gradually decline and then display a gradual increase through 11.5 hour postoviposition.;M. domestica anti-ODC specific antibodies were developed and used in western blot analysis of developing embryos collected at 30-minute intervals. Protein levels corresponded to the presence of the ODC mRNA. One difference was the detection of ODC protein during 0 to 1.0 hour postoviposition, which likely represents maternally expressed ODC protein. Between 1.5 and 6.0 hour postoviposition, the ODC protein is completely undetectable until the zygotic ODC protein appears at 6.5 to 7.0 hour postoviposition. ODC protein levels remain relatively unchanged until 11.5 hour postoviposition when they decrease to nearly undetectable levels.;However, ODC activity was nearly undetectable until 9 hour postoviposition, with only a small increase in activity through 10.5 hour postoviposition. At 11.0 hour postoviposition, a dramatic increase in ODC activity was observed, followed by a dramatic decrease at 11.5 hour postoviposition.;These data demonstrate that M. domestica ODC is regulated at both the level of transcription and the level of translation. Additionally, these data indicate that ODC is also regulated at the post-translation level and that this regulation may be related to the expression of antizyme mRNA.