Gene trap mutagenesis-based forward genetic approach reveals that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2.
OVCA1 is a tumor suppressor identified by positional cloning from chromosome 17p13.3, a hot spot for chromosomal aberration in breast and ovarian cancers. It has been shown that expression of OVCA1 is reduced in some tumors and that it regulates cell proliferation, embryonic development, and tumorigenesis. However, the biochemical function of OVCA1 has remained unknown. Recently, we isolated a novel mutant resistant to diphtheria toxin and Pseudomonas exotoxin A from the gene trap insertional mutants library of Chinese hamster ovary cells. In this mutant, the Ovca1 gene was disrupted by gene trap mutagenesis, and this disruption well correlated with the toxin-resistant phenotype. We demonstrated direct evidence that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2, the target of bacterial ADP-ribosylating toxins. A functional genetic approach utilizing the random gene trap mutants library of mammalian cells should become a useful strategy to identify the genes responsible for specific phenotypes.
Nobukuni
Yoshitaka
Y
Department of Human Genetics, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. nobukuni@hiroshima-u.ac.jp
Kohno
Kenji
K
Miyagawa
Kiyoshi
K
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D016466:CHO Cells; D004396:Coloring Agents / Q000494:pharmacology; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004167:Diphtheria Toxin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D016147:Genes, Tumor Suppressor; D005821:Genetic Techniques; D006639:Histidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D020652:Peptide Elongation Factor 2 / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014118:Toxins, Biological; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D004396:Coloring Agents; D018076:DNA, Complementary; C121987:DPH1 protein, human; C115726:DPH2 protein, human; D004167:Diphtheria Toxin; D015778:Minor Histocompatibility Antigens; D020652:Peptide Elongation Factor 2; D011506:Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D014118:Toxins, Biological; D025521:Tumor Suppressor Proteins; D006639:Histidine; D000244:Adenosine Diphosphate; C027527:diphthamide; C022616:thiazolyl blue