Related Experiment Videos
Overexpression of wild-type and mutant NDP kinase in Dictyostelium discoideum
O Sellam1, M Véron, M Hildebrandt
1Unité de Biochimie Cellulaire, CNRS, URA1129, Institut Pasteur, Paris, France.
Abstract:
Nucleoside diphosphate (NDP) kinase has a central role in the synthesis of (deoxy-)trinucleotides. In addition, mutations in the gene encoding NDP kinase have been shown to have important consequences for Drosophila development and mammalian tumorogenesis. We have overexpressed, in Dictyostelium discoideum, a genomic clone encoding the enzyme NDP kinase. The concomitant increase in the levels of RNA and enzyme activity identifies a 5' non-coding genomic region of 0.9 kb as being the complete promoter region. Overexpression of wild-type NDP kinase has no effect on development. This is also true for an inactive mutant H122C that does not have a dominant inhibitor effect. Overexpression of the P105G mutant NDP kinase, which is known to be affected in its stability in vitro, only leads to a small increase in total NDP-kinase activity. Thermal and chemical denaturation experiments demonstrate the formation of hexameric hybrids between wild-type and mutant monomers.
Insights
Nucleoside diphosphate (NDP) kinase is crucial for nucleotide synthesis. Overexpressing NDP kinase in Dictyostelium discoideum identified its promoter region but did not affect development, even with mutants.
Area of Science:
- Molecular Biology
- Biochemistry
- Developmental Biology
Background:
- Nucleoside diphosphate (NDP) kinase is essential for (deoxy-)trinucleotide synthesis.
- NDP kinase gene mutations impact Drosophila development and mammalian tumorogenesis.
Purpose of the Study:
- To overexpress NDP kinase in Dictyostelium discoideum to identify its promoter region.
- To investigate the developmental effects of wild-type and mutant NDP kinase overexpression.
Main Methods:
- Overexpression of a genomic clone encoding NDP kinase in Dictyostelium discoideum.
- RNA and enzyme activity level analysis.
- Thermal and chemical denaturation experiments to study protein stability.
Main Results:
- A 0.9 kb 5' non-coding genomic region was identified as the complete promoter for NDP kinase.
- Overexpression of wild-type NDP kinase and an inactive mutant (H122C) had no effect on development.
- Overexpression of a stability-affected mutant (P105G) resulted in a minor increase in total NDP-kinase activity.
- Hexameric hybrids formed between wild-type and mutant monomers upon denaturation.
Conclusions:
- The 5' non-coding region of 0.9 kb is the complete promoter for NDP kinase.
- NDP kinase activity levels do not appear to be a rate-limiting factor for Dictyostelium development under these conditions.
- Mutant NDP kinase monomers can form hybrid hexamers with wild-type monomers, suggesting potential dominant-negative or altered activity mechanisms.