Recombinant ATM protein complements the cellular A-T phenotype

摘要: Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability and radiation sensitivity. The cellular phenotype of A-T points to defects in signal transduction pathways involved activation cell cycle checkpoints free radical damage, other that mediate the transmission specific mitogenic stimuli. product responsible gene, ATM, belongs a family large proteins contribute maintaining stability progression various organisms. A recombinant vector stably expresses full-length ATM protein valuable tool for its functional analysis. We constructed cloned recombinant, open reading frame using combination vectors hosts overcame inherent this sequence. Recombinant was expressed insect cells baculovirus vector, albeit at low level, human episomal expression vector. An amino-terminal FLAG epitope added allowed highly detection molecule immunoblotting, immunoprecipitation immunostaining, isolation immunoaffinity. Similar endogenous located mainly nucleus, with levels cytoplasm. Ectopic restored normal sensitivity ionizing radiomimetic drug neocarzinostatin, pattern post-irradiation DNA synthesis, which represents S-phase checkpoint. These observations indicate epitope-tagged functional. Introduction into known missense mutation, Glu2904Gly, resulted apparent inability complement phenotype. findings physiological characteristic result from absence protein, deficiency can be corrected ectopic protein.