Isolation and characterization of Xenopus ATM (X-ATM): expression, localization, and complex formation during oogenesis and early development

ATM, the gene product mutated in Ataxia Telangiectasia (A-T) encodes a 350- KDa protein involved in the regulation of several cellular responses to DNA breaks. We used a degenerate PCR-based strategy to isolate a partial clone of X-ATM, the Xenopus homologue of human ATM. Sequence analysis and confir med that the clone was most closely related to human ATM. Xenopus ATM prote in (X-ATM) is 85% identical to human ATM within the kinase domain and 71% i dentical over the carboxyl-terminal half of the protein. Polyclonal antibod ies raised against recombinant Ii-ATM are highly specific for the ATM prote in and recognize a single polypeptide of 370-kDa in oocytes, embryos, egg e xtracts and a,Xenopus cell line. We found that X-ATM, mas expressed materna lly in eggs and as early as stage II pre-vitellogenic oocytes, and the prot ein and mRNA sere present at relatively constant levels throughout developm ent. Subcellular fractionation showed that the protein mas nuclear in both the female and male germlines. The level of X-ATM protein did not change th roughout the meiotic divisions or the synchronous mitotic cycles of cleavag e stage embryos. In addition, we did not observe any change in the level or mobility of X-ATM protein following ii-irradiation of embryos. Finally, we also demonstrated that X-ATM was present in a high molecular weight comple x of approximately 500 kDa containing the X-ATM protein and other, as yet u nidentified component(s).