Molecular Cancer Research 2:348-353 (2004)
© 2004 American Association for Cancer Research
Cancer Genes and Genomics
Molecular Cloning and Characterization of the Catalytic Domain of Zebrafish Homologue of the Ataxia-Telangiectasia Mutated Gene1
Renu Garg1,
Chuan-Dong Geng2,
Jennifer L. Miller3,
Shannon Callens1,
Xi Tang1,
Bruce Appel3 and
Bo Xu1,2
1 Department of Genetics and Stanley S. Scott Cancer Center, LSU Health Sciences Center, New Orleans, Louisiana;
2 Department of Biochemistry and Molecular Biology, LSU Health Sciences Center, New Orleans, Louisiana; and
3 Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee
Requests for reprints: Bo Xu, Department of Genetics and Stanley S. Scott Cancer Center, LSU Health Sciences Center, Room 406 CSRB Building, 533 Bolivar Street, New Orleans, LA 70112. Phone: (504) 568-2228; Fax: (504) 568-8500. E-mail: bxu{at}lsuhsc.edu
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Abstract
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Inherited biallelic mutations of the ATM (ataxia-telangiectasia mutated) gene in humans cause ataxia-telangiectasia, a rare autosomal recessive disorder associated with progressive neuro-degeneration, cancer predisposition, immunodeficiency, and hypersensitivity to ionizing radiation. The ATM gene is highly conserved across a wide range of species. In an attempt to establish a zebrafish (Danio rerio) model of ataxia-telangiectasia, we cloned the coding sequence of the catalytic domain of the zebrafish homologue of ATM and found it to contain an open reading frame encoding 907 amino acids at the carboxyl terminus of the zebrafish ATM (zATM). The catalytic domain of zATM shares 67% and 66% homology with human ATM (hATM) and mouse ATM (mATM), respectively. The full-length mRNA encoding zATM is found to be approximately 11 kb by Northern hybridization, and the expression of zATM is observed in different adult and embryonic tissues. Overexpression of a kinase-inactive zATM domain in human cells has a dominant-negative effect against hATM function. Expression of the altered zATM in ZF4 cells leads to an A-T–like phenotype in response to ionizing radiation. These results taken together indicate that zATM is the homologue of hATM. Furthermore, using the kinase-inactive form of zATM should allow manipulation of zATM function in fish cells.
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Introduction
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The human autosomal recessive disease ataxia-telangiectasia (A-T) is characterized by cerebellar ataxia, resulting from progressive neuro-degeneration, and telangiectasia, dilation of blood vessels within the eyes and parts of the facial region (1). A-T homozygotes suffer from recurrent infections caused by immune deficiencies and as a population, have an approximately 5-fold increased risk of developing lymphomas and leukemias. Early attempts at treating these malignancies with radiotherapy revealed another hallmark of A-T: a profound hypersensitivity to the cytotoxic effects of ionizing radiation (IR). Cells derived from A-T patients display a complex phenotype, including chromosomal instability, defective cell cycle checkpoints, and hypersensitivity to IR and radiomimetic drugs.
Despite the rarity of the disease (affecting approximately 1 of 40,000 to 1 of 100,000), interest in the function of the gene product (ATM) is intense because of the complexity of clinical and cellular phenotypes of germline mutation of the gene. The gene mutated in A-T, ATM, encodes a Mr 370,000 protein that is remarkable for its large size and the existence of a sequence in its carboxyl terminus similar to phosphatidylinositol 3 (PI-3) kinases (2, 3). A family of proteins, including Tel1, Mec1, and Rad3 in yeast, Mei-41 in Drosophila, and ATR and DNA-PK in vertebrates, is well conserved and has similar large sizes and carboxyl-terminal kinase sequences, all of which are critical in DNA damage responses (4). The well-established role of ATM is in regulation of IR-induced cell cycle checkpoints (4, 5), the lack of which may contribute to genetic instability and cancer formation (6). In unperturbed cells, ATM is held inactive as a dimer or higher-order multimer, with the kinase domain bound to an internal domain of a neighboring ATM molecule. In response to DNA double-strand breaks (DSB), ATM dimers separate when they phosphorylate each other at a particular amino acid serine (7). The monomer form of ATM becomes activated and it phosphorylates a series of targets to activate cell cycle checkpoints (5, 8). The critical role of ATM in DNA damage response is also implicated in limiting neurologic abnormalities. In the developing nervous system, ATM is required to eliminate neural cells that have incurred DNA damage (9-12).
Among current model systems that have been developed for studying the functional role of ATM, the mouse system is the only vertebrate model system that has been used successfully. However, though ATM-null mice are radiosensitive, cancer prone, and sterile, they do not show neuro-degenerative phenotype (13). These mice succumb to lymphoma from an early age, but can live past 1 year in the absence of tumors. Because of considerable progress in zebrafish (Danio rerio) genetics and genomics (14), the zebrafish system can be an alternate vertebrate model for studying the functional role of ATM on neurodevelopment and cancer genetics, and for screening ATM inhibitors as radiosensitizers. However, no clinical entity corresponding to A-T has been described to date in this system. We report here that the catalytic domain of zebrafish ATM (zATM) shares high homology with human ATM (hATM) and mouse ATM (mATM). Expression of a kinase-inactive zATM domain causes A-T–like cellular phenotype in zebrafish cells and has a dominant-negative effect against hATM.
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Results and Discussion
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Molecular Cloning of the Catalytic Domain of zATM
To search for a cDNA clone from zebrafish corresponding to the hATM, a zebrafish embryo cDNA library was screened at a moderate stringency with a PCR-generated DNA probe corresponding to the PI-3 kinase domain of hATM cDNA. Three positive clones were identified and sequenced. All three of the clones contained identical nucleotide sequences but differed in the length of cDNA insert; the longest clone contained a nucleotide sequence of 3.19 kb (GenBank accession no. AJ605775). This 3.19-kb cDNA fragment encoded an open reading frame of 907 amino acids followed by a stop codon, suggesting this to be the carboxyl terminus of the putative protein. The 3' untranslated region was about 456 bp long followed by a poly(A) tail. The nucleotide sequence of the positive clone was used to search for the homology using BLAST-X server, and was found to have homology with ATM from different organisms (Table 1). The maximum homology was found with the hATM (67%) and mATM (66%) sequences at the protein level. The comparison of the carboxyl-terminal sequences spanning the PI-3 kinase domain of hATM and mATM with that of zATM revealed that zATM is 79% and 78% identical to the hATM and mATM, respectively, in this region (Fig. 1), whereas the hATM and mATM sequences matched 92% to each other in this region. The PI-3 kinase domain signature sequence (15) was identical in zATM, mATM, and hATM. However, there was only 15% similarity between identified zATM and human ATR sequences (Table 1). These results indicate that the sequence we have identified is indeed the kinase domain of the zebrafish homologue of hATM.
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FIGURE 1. Alignment of the carboxyl-terminal sequence of hATM, mATM, and zATM. The amino acid sequences from the carboxyl-terminal ends of hATM, mATM, and zATM were aligned for the best match using the CLUSTAL W program. Asterisk, sequence identical in all three species; period or semicolon, sequence similar in all three species. The PI-3 kinase signature sequence is shown in boldface.
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Expression of zATM in Adult Tissues
To test the expression of zATM and to estimate the size of the full-length zATM mRNA, we did the Northern blot hybridization and reverse transcription (RT)-PCR using the total RNA from various adult zebrafish tissues. The Northern blot hybridization of total RNA from zebrafish eyes using the 3-kb zATM cDNA probe identified a message of about 11 kb (Fig. 2A), suggesting that the cloned zATM fragment represents only a part of the full-length zATM cDNA. This is consistent with similar large sizes of the proteins in the ATM family. The RT-PCR analysis revealed the expression of zATM in several zebrafish tissues, including eye, brain, oocyte, and intestine, suggesting that zATM is expressed ubiquitously in all of these adult tissues (Fig. 2B and C).
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FIGURE 2. Expression of zATM in adult tissues from zebrafish. A. Northern blot of total RNA from zebrafish eyes probed with a labeled 3-kb zATM fragment. The RNA marker lane was hybridized with the labeled  HindIII DNA. B. Representative gel photographs of the RT-PCR products using RNA from the indicated zebrafish tissues and the zATM or zActin specific PCR primers. C. The above RT-PCR products were blotted onto nylon membrane and probed with labeled zATM cDNA. The autoradiogram of zATM specific RT-PCR products is shown, whereas the RT-PCR products obtained from zActin specific primers did not yield any signal using the above probe.
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Expression of zATM in Embryonic Tissues
To examine the distribution of ATM transcripts in embryos, we did in situ RNA hybridization at several developmental time points using digoxygenin-labeled antisense probes. At neural plate stage (10.5 to 11 hours post-fertilization), ATM transcripts were distributed uniformly at a low level throughout the embryo (data not shown). At 24 hours-post-fertilization (hpf), the eye and ventricular regions of ventral diencephelon, midbrain, and cerebellum showed elevated levels of ATM expression (Fig. 3). By 48 hpf, low levels of ATM were evident in ventricular regions of the brain (data not shown). It is noted that a sense probe control for the experiments shows a negative response (data not shown).
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FIGURE 3. Expression of zATM in embryos. Shown are in situ hybridization analyses of zATM RNA in 24 hpf: (A) lateral view with anterior to the left; (B) dorsal view with anterior to the left; and (C) cross section through the forebrain and eyes. Abbreviations: ce, cerebellum; di, diencephalon; ey, eye; hi, hindbrain; mi, midbrain; and te, telencephalon.
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Construction and Expression of the Catalytic Domain of zATM Into Human and Zebrafish Cells
To further characterize zATM, the catalytic domain of zATM (herein called zATMp) was subcloned into a mammalian expression vector pCDNA3. A mutant form of the zATMp cDNA was also engineered, which contained two putative kinase-inactive mutations (Asp719
Ala and Asn724Lys) based on the similar mutations resulting in a kinase-inactive hATM (16). The wild-type (wt-) and kinase-inactive (ki-) cDNAs of zATMp were tagged with an amino-terminal flag epitope. Expression of these constructs in 293T and ZF4 (a zebrafish cell line) cells is shown in Figs. 4A and 5A. In vitro kinase assays using glutathione S-transferase (GST)–p53 (amino acids 1 to 101) as a substrate of zATMp confirmed the existence of kinase activity of wt-zATMp and a decreased kinase activity in ki-zATMp (data not shown).
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FIGURE 4. Overexpression of a ki-zATMp inhibits hATM function. 293T cells transiently transfected with empty vector, wild-type (wtATM) or kinase-dead (kdATM) hATM, or wild-type (wt-zATMp) or kinase inactive (ki-zATMp) form of zATMp were assessed for: (A) expression of transduced flag-tagged ATMs by immunoblotting with an anti-flag antibody; (B) replicative DNA synthesis 30 minutes after 10 Gy of IR; and (C) IR-induced G2-M checkpoint: The mitotic percentage change of 293T cells 90 minutes after 6 Gy of IR is shown. Mitotic cell percentage was determined by anti-phospho-histone H3 staining followed by flow cytometric analysis. Error bars, averages of at least triplicate samples.
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FIGURE 5. Overexpression of the ki-zATMp results in an A-T–like phenotype in ZF4 cells. ZF4 cells transiently transfected with empty vector, wild-type (wt-zATMp), or kinase-inactive (ki-zATMp) form of zATMp were assessed for: (A) expression of transduced flag-tagged ATMs by immunoblotting with an anti-flag antibody; and (B) replicative DNA synthesis 30 minutes after 10 Gy of IR. Error bars, averages of at least triplicate samples.
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Expression of the Kinase-Inactive Form of zATMp in Human Cells Results in a Dominant-Negative Inhibition of hATM Function
Because the catalytic domain of zATM shares high homology with hATM, we hypothesized that expressing the kinase-inactive form of zATMp would inhibit hATM function in vivo. To test this note, we expressed wt- or ki-zATMp into 293T cells and assessed IR-induced S-phase and G2-M checkpoints, both of which are dependent on the hATM function (17, 18). The IR-induced S-phase checkpoint was assessed by measuring replicative DNA synthesis after IR (19). We find that expression of ki-zATMp abrogates the S-phase checkpoint as we see less decrease of DNA synthesis compared with vector- or wt-zATMp–transfected cells after 10 Gy of IR (Fig. 4B). As a control, expressing the kinase-dead (kd) hATM also results in defective S-phase arrest in 293T cells. It is noted that expression of zATMp does not change the basal level of DNA synthesis in cells. The IR-induced G2-M checkpoint was assessed by counterstaining phospho-histone H3 and propidium iodide followed by flow cytometric analysis (20). 293T cells have a normal G2-M checkpoint, showing transient inhibition of mitotic entry after IR. We observed that cells expressing ki-zATMp fail to arrest at G2 compared with vector or wt-zATMp–transfected partners after 6 Gy of IR (Fig. 4C). These data taken together suggest that expressing the kinase-inactive form of zATMp has a dominant-negative effect on hATM function. This dominant-negative effect is also observed in HeLa cells (data not shown). These observations further support that zATM is a bona fide homologue of hATM.
Expression of the Kinase-Inactive Form of zATMp Abrogates ATM-Mediated S-Phase Checkpoint in ZF4 Cells
To develop ways to manipulate zATM function in zebrafish cell lines, we examined the ability of ki-zATMp to exhibit dominant-negative activity in ZF4 cells by looking at the IR-induced S-phase checkpoint. ZF4 is a fibroblast cell line derived from zebrafish embryo tissues. It displays a normal response as shown by inhibition of DNA synthesis in irradiated cells relative to unirradiated cells at 30 minutes after IR (Fig. 5B, ZF4). Following 10 Gy of IR, the mock-treated ZF4 cells exhibit an approximately 60% decrease in DNA synthesis. Expressing vector only or wt-zATMp does not affect the S-phase checkpoint (Fig. 5B). In contrast, cells expressing ki-zATMp exhibit a lack of inhibition of DNA synthesis in response to IR. These results indicate that the S-phase checkpoint in ZF4 cells can be abrogated by expressing the kinase-inactive form of zATMp, suggesting that the altered zATMp functions dominant negatively by competing with endogenous zATM protein in binding to its normal cellular partners.
In summary, we cloned the catalytic domain of zATM and assessed the expression pattern of this gene product in adult and embryonic tissues. We find that zATMp shares high homology with hATM and mATM. We also find that overexpressing a ki-zATMp recapitulated an A-T–like phenotype in zebrafish cells. Use of this altered construct should now permit manipulation of zATM function in transfectable zebrafish cells, and furthermore, should provide a useful tool for establishing a zebrafish model of A-T.
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Materials and Methods
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Library Screening and Sequence Analysis
About 1 x 106 recombinants were screened from a cDNA library constructed from 15 to 19 hours post-fertilization zebrafish embryo poly(A)+ RNA using Uni-ZAP XR vector (Stratagene, La Jolla, CA). A 0.88-kb DNA probe (nt 8470 to 9354 of hATM cDNA, accession no. U33841) was generated by PCR and labeled with horseradish peroxidase using enhanced chemiluminescence (ECL) Direct Nucleic Acid labeling and detection system (Amersham Biosciences, Piscataway, NJ). The library was plated at a density of 5 x 104 plaque-forming unit per plate and the plaques were lifted onto the hybond N+ membrane using standard procedures. The membrane filters were pre-hybridized in gold hybridization buffer (provided with the kit) for 2 hours at 42°C. The hybridization was done at 38°C for 16 to 18 hours in the same buffer containing 10 ng/mL labeled probe. The filters were washed twice with 0.5x SSC, 0.4% SDS, and 6 mol/L urea at 38°C for 20 minutes each and rinsed with 2x SSC. The detection was carried out according to the manufacturer's instructions. The positive clones were plaque purified and bluescript SK– plasmids were excised from the vector using ExAssist helper phage. The inserts of all the positive clones were fully sequenced and the homology search was done at the nucleotide and amino acid level using BLAST server.
Northern Blot Hybridization and RT-PCR Analysis
The total RNA was extracted from zebrafish tissue using Trizol reagent (Invitrogen, Carlsbad, CA) and about 20 µg of total RNA were subjected to agarose gel electrophoresis followed by blotting onto the nylon membrane. The membrane was probed with the 3-kb zATM probe labeled with horseradish peroxidase as described above. For the RT-PCR analysis, about 5 µg of total RNA from different tissues of zebrafish were reverse transcribed using Superscript II reverse transcriptase (Invitrogen) primed with oligodeoxythimidylate [oligo(dT)] primer. The generated cDNA was used as the template for PCR using either zATM or zActin specific primers and platinum Taq DNA polymerase (Invitrogen). Control experiments were done with RNA samples but without reverse transcriptase. The PCR products were electrophoresed through 1.5% agarose gel, blotted onto the nylon membrane, and hybridized with labeled zATM probe to confirm their identity.
In situ RNA Hybridization
In situ RNA hybridization was done essentially as previously described (21). Following the color reaction, embryos were dissected from the yolk and mounted on microscope slides in 75% glycerol. Images were obtained using a QImaging Retiga Exi color CCD camera mounted on a compound microscope. Images were imported into Adobe Photoshop. Image manipulations were limited to levels, curves, hue, and saturation adjustments.
Mammalian Expression Plasmids
Fragments of DNA encoding zATM were amplified with Pfu polymerase and were epitope tagged by subcloning the fragments into pcDNA3 (Invitrogen) that had flag-epitope tag inserted. The Quick-Change Site-Directed Mutagenesis kit (Stratagene) was used to generate the ki-zATMp.
Cell Lines and Delivery of Radiation
Human 293T cells (American Type Culture Collection, Manassas, VA) were grown as monolayers in DMEM supplemented with 10% fetal bovine serum. They were grown at 37°C in a humidified atmosphere containing 5% CO2. Zebrafish cell line ZF4 (American Type Culture Collection) was cultured in 1:1 mixture of Ham's F12 and DMEM containing 10% fetal bovine serum and 100 units/mL penicillin/streptomycin (pen/strep). They were grown at 28°C in a humidified atmosphere containing 5% CO2. IR was delivered from a 137Cs source at a rate of approximately 120 cGy/min.
Transfection Experiments
Transfection of wild-type hATM, kinase-dead hATM (both were provided by Dr. Michael B. Kastan, St. Jude Children's Research Hospital, Memphis, TN), wild-type zATMp, and kinase-inactive zATMp were done in the logarithmic phase of growth with LipofectAMINE (Invitrogen). The efficiency of transfection was assessed by transfection with a green fluorescent protein (GFP) reporter vector and analyzing for green fluorescent protein expression by flow cytometry 36 hours after transfection. The efficiency of transfection in multiple assessments was always between 70% and 90% (data not shown).
Western Blot Analyses
To detect expression of transfected ATMs, cells were harvested 36 hours after transfection, and resuspended in TGN lysis buffer. Protein concentrations were determined using the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA). Samples containing equal amounts of protein were mixed with an equal volume of 2x Laemmli sample buffer, boiled, and separated by SDS-PAGE. Proteins were transferred to nitrocellulose and immunoblotted with an anti-flag M5 antibody (Sigma Chemical Co., St. Louis, MO).
The S-Phase Checkpoint Assay
IR-induced inhibition of DNA synthesis was assessed as described previously (18). Cells were pre-labeled by culturing them in DMEM that contained 10 nCi/mL of [14C]thymidine (NEN Life Science Products, Inc., Boston, MA) for 
24 hours after transfection. The medium containing [14C]thymidine was then replaced with normal DMEM, and the cells were incubated for 6 hours. Cells were irradiated with 10 Gy of IR. Thirty minutes after IR, cells were pulse-labeled with 2.5 µCi/mL [3H]thymidine for 15 minutes (NEN). Cells were harvested, washed twice with PBS, and fixed in 70% methanol for at least 30 minutes. After the cells were transferred to Whatman filters and fixed sequentially with 70% methanol and then with 95% methanol, the amount of radioactivity on the filter was assayed in a liquid scintillation counter. The measure of DNA synthesis was derived from resulting ratios of 3H counts/min to 14C counts/min, corrected for those counts per minute that resulted from channel crossover.
The G2-M Checkpoint Assay
Cells were harvested 90 minutes after 6 Gy of IR and fixed in 70% ethanol at –20°C. The cells were suspended in 100 µL of PBS containing 1% bovine serum albumin and 0.75 µg of a polyclonal antibody that specifically recognizes the phosphorylated form of histone H3 (Upstate Biotechnology, Lake Placid, NY) and incubated for 3 hours at room temperature. The cells were then rinsed with PBS containing 1% bovine serum albumin and incubated with FITC-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted at a ratio of 1:30 in PBS containing 1% bovine serum albumin. After 30 minutes of incubation at room temperature in the dark, the cells were stained with propidium iodide (Sigma), and cellular fluorescence was measured by a FACSCalibur flow cytometer.
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Acknowledgements
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We thank Dr. Michael B. Kastan (St. Jude Children's Research Hospital) for providing wild-type and kinase-dead human ATM constructs. We also thank Dr. Oliver Wessely (Department of Cell Biology, LSUHSC) and all members in the Xu laboratory for helpful discussions.
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Notes
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1 Note: Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession no. AJ605775. 
Received March 4, 2004;
revised May 4, 2004;
accepted May 14, 2004.
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References
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- Shiloh Y. Ataxia-telangiectasia and the Nijmegen breakage syndrome: related disorders but genes apart. Annu Rev Genet 1997;31:635-62.[CrossRef][Medline]
- Savitsky K, Bar-Shira A, Gilad S, et al. A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science 1995;268:1749-53.[Abstract/Free Full Text]
- Savitsky K, Sfez S, Tagle DA, et al. The complete sequence of the coding region of the ATM gene reveals similarity to cell cycle regulators in different species. Hum Mol Genet 1995;4:2025-32.[Abstract/Free Full Text]
- Abraham RT. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 2001;15:2177-96.[Free Full Text]
- Kastan MB, Lim D-S. The many substrates and functions of ATM. Nat Rev Mol Cell Biol 2000;1:179-86.[CrossRef][Medline]
- Elledge SJ. Cell cycle checkpoints: preventing and identity crisis. Science 1996;274:1664-72.[Abstract/Free Full Text]
- Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 2003;421:499-506.[CrossRef][Medline]
- Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 2003;3:155-68.[CrossRef][Medline]
- Allen DM, van Praag H, Ray J, et al. Ataxia telangiectasia mutated is essential during adult neurogenesis. Genes Dev 2001;15:554-66.[Abstract/Free Full Text]
- Lee Y, Chong MJ, McKinnon PJ. Ataxia telangiectasia mutated-dependent apoptosis after genotoxic stress in the developing nervous system is determined by cellular differentiation status. J Neurosci 2001;21:6687-93.[Abstract/Free Full Text]
- Lee Y, McKinnon PJ. ATM dependent apoptosis in the nervous system. Apoptosis 2000;5:523-9.[CrossRef][Medline]
- Miller HL, Lee Y, Zhao J, Chong MJ, McKinnon PJ. Atm and c-Abl cooperate in the response to genotoxic stress during nervous system development. Brain Res Dev Brain Res 2003;145:31-8.[Medline]
- McKinnon PJ. Ataxia telangiectasia: new neurons and ATM. Trends Mol Med 2001;7:233-4.[CrossRef][Medline]
- Stern HM, Zon LI. Cancer genetics and drug discovery in the zebrafish. Nat Rev Cancer 2003;3:533-9.[CrossRef][Medline]
- Pecker I, Avraham KB, Gilbert DJ, et al. Identification and chromosomal localization of Atm, the mouse homolog of the ataxia-telangiectasia gene. Genomics 1996;35:39-45.[CrossRef][Medline]
- Canman CE, Lim D-S, Cimprich KA, et al. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 1998;281:1677-9.[Abstract/Free Full Text]
- Lim D-S, Kim S-T, Xu B, et al. ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway. Nature 2000;404:613-7.[CrossRef][Medline]
- Xu B, Kim S-T, Kastan MB. Involvement of Brca1 in S-phase and G2-phase checkpoints after ionizing irradiation. Mol Cell Biol 2001;21:3445-50.[Abstract/Free Full Text]
- Painter RB, Young BR. Radiosensitivity in ataxia-telangiectasia: a new explanation. Proc Natl Acad Sci 1980;77:7315-7.[Abstract/Free Full Text]
- Xu B, Kim ST, Lim DS, Kastan MB. Two molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation. Mol Cell Biol 2002;22:1049-59.[Abstract/Free Full Text]
- Hauptmann G, Gerster T. Multicolor whole-mount in situ hybridization. Methods Mol Biol 2000;137:139-48.[Medline]
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Abstract
Introduction
