Overexpression of wild-type and mutant NDP kinase in Dictyostelium discoideum.
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.
Sellam
O
O
Unité de Biochimie Cellulaire, CNRS, URA1129, Institut Pasteur, Paris, France.
Véron
M
M
Hildebrandt
M
M
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D004023:Dictyostelium / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000096:biosynthesis; D018507:Gene Expression Regulation, Developmental; D009154:Mutation; D009701:Nucleoside-Diphosphate Kinase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010957:Plasmids; D011487:Protein Conformation; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013696:Temperature; D013997:Time Factors; D014170:Transformation, Genetic
D005656:Fungal Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D009701:Nucleoside-Diphosphate Kinase