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Related Experiment Videos

Recombinant ATM protein complements the cellular A-T phenotype

Y Ziv1, A Bar-Shira, I Pecker

  • 1Department of Human Genetics, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel.

Oncogene
|July 10, 1997
PubMed
Summary

Ataxia-telangiectasia (A-T) is a genetic disorder. Scientists created a functional ATM protein to correct A-T cell defects, restoring normal radiation sensitivity and DNA synthesis.

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Area of Science:

  • Genetics and Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Ataxia-telangiectasia (A-T) is an autosomal recessive disorder with neurodegeneration, immunodeficiency, and cancer predisposition.
  • Cellular defects in A-T involve signal transduction pathways crucial for cell cycle checkpoints and response to DNA damage.
  • The ATM gene product is a large protein family member essential for genome stability and cell cycle progression.

Purpose of the Study:

  • To construct and analyze a full-length recombinant ATM protein for functional studies.
  • To develop a tool for investigating ATM protein function in Ataxia-telangiectasia.
  • To assess the ability of recombinant ATM to correct the cellular phenotype of A-T cells.

Main Methods:

  • Constructed and cloned a full-length ATM open reading frame using a combination of vectors and hosts.

Related Experiment Videos

  • Expressed recombinant ATM in insect cells via baculovirus vector and in human A-T cells using an episomal vector.
  • Utilized an N-terminal FLAG epitope for detection, isolation, and functional analysis of recombinant ATM.

    • Main Results:

    • Recombinant ATM was stably expressed in both insect and human A-T cells.
    • Ectopic expression of ATM in A-T cells restored normal ionizing radiation sensitivity and post-irradiation DNA synthesis.
    • A specific A-T missense mutation (Glu2904Gly) in recombinant ATM led to protein instability and failed to complement the A-T phenotype.

    Conclusions:

    • The recombinant, epitope-tagged ATM protein is functional and can correct the cellular defects in Ataxia-telangiectasia.
    • The physiological defects in A-T cells are due to the absence of functional ATM protein.
    • Ectopic expression of ATM can correct the deficiency characteristic of Ataxia-telangiectasia cells.