Abstract
Neuronal degeneration, gonadal abnormalities, and immune deficiency are some of the major manifestations of the hereditary disease ataxia telangiectasia, which is caused by mutations in a single gene, designated ATM. Here we show that the product of the ATM gene is a 370-kDa nuclear phosphoprotein. Because ATM knockout mice recapitulate the clinical symptoms of the human disease, we have examined ATM gene expression in mice. In mouse embryos at gestation day 13.5, ATM mRNA is expressed ubiquitously, with high levels detected in the nervous system and lung. Elevated ATM mRNA levels were also found in the thymus of mouse embryos at gestation day 18.5, a time when V(D)J recombination is occurring. In adult mice, ATM protein was detected in all tissues examined and was present at elevated levels in the testis, spleen, and thymus. The ATM expression pattern and the nuclear localization of the ATM protein are consistent with the proposed function of ATM in the activation of cell cycle checkpoints, DNA repair, and genetic recombination.
Highlights
Ataxia telangiectasia (AT)1 is a rare autosomal recessive disease that affects multiple organs [1]
By using two antisera raised against different parts of ATM, we identified the ATM gene product as a protein of 370 kDa, a molecular mass that is close to the calculated molecular weight of 350,000 from the ATM cDNA [16]
The specific mutations of ATM in the three AT cell lines are unknown, earlier studies indicate that most ATM mutations in individuals with AT would result in mutant ATM gene products with large deletions or truncations [31, 32], which are unlikely to be detected by an antiserum raised against the C terminus of ATM
Summary
Antibodies and Cell Lines—Antibodies against DNA-PKcs [20] were kindly provided by Tom Shenk (Princeton University). Production of Anti-ATM Antibodies—Reverse transcription-polymerase chain reaction was performed to obtain two cDNAs encompassing human ATM cDNA sequences 2940 – 4536 and 7731–9168, respectively. After confirmation of their identity with ATM sequence [17] by DNA. Mouse polyclonal antisera specific for the fusion proteins and hyridoma cell lines were generated according to standard procedures [22]. Immunoprecipitations, including all washes, were performed with EBC buffer as described previously [24]. After washing with phosphate-buffered saline, the cells were permeabilized with 0.3% Triton in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, and 0.05% Tween 20) for 10 min. In situ hybridization was performed essentially as described by Cox et al [26]
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