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

Oncogene volume 18, pages 7070–7079 (1999)Cite this article

Abstract

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 confirmed that the clone was most closely related to human ATM. Xenopus ATM protein (X-ATM) is 85% identical to human ATM within the kinase domain and 71% identical over the carboxyl-terminal half of the protein. Polyclonal antibodies raised against recombinant X-ATM are highly specific for the ATM protein and recognize a single polypeptide of 370-kDa in oocytes, embryos, egg extracts and a Xenopus cell line. We found that X-ATM was expressed maternally in eggs and as early as stage II pre-vitellogenic oocytes, and the protein and mRNA were present at relatively constant levels throughout development. Subcellular fractionation showed that the protein was nuclear in both the female and male germlines. The level of X-ATM protein did not change throughout the meiotic divisions or the synchronous mitotic cycles of cleavage stage embryos. In addition, we did not observe any change in the level or mobility of X-ATM protein following γ-irradiation of embryos. Finally, we also demonstrated that X-ATM was present in a high molecular weight complex of approximately 500 kDa containing the X-ATM protein and other, as yet unidentified component(s).

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Acknowledgements

We would like to thank Dr Yen for the gift of the human ATM antibody used in this study. This work is supported by US Army grants DAMD 17-97-2-7075 to C Hensey and DAMD 17-97-1-7071 and the Mallinkcrodt Foundation to J Gautier.

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Authors and Affiliations

  1. Department of Genetics and Development, College of Physicians and Surgeons of Columbia University, 630 West 168th Street, New York, 10032, NY, USA

    Kirsten Robertson, Carmel Hensey & Jean Gautier

  2. Department of Dermatology, College of Physicians and Surgeons of Columbia University, 630 West 168th Street, New York, 10032, NY, USA

    Kirsten Robertson, Carmel Hensey & Jean Gautier

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Robertson, K., Hensey, C. & Gautier, J. Isolation and characterization of Xenopus ATM (X-ATM): expression, localization, and complex formation during oogenesis and early development. Oncogene 18, 7070–7079 (1999). https://doi.org/10.1038/sj.onc.1203194

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