Showing papers by "Yosef Shiloh published in 1997"

The genetic defect in ataxia-telangiectasia

Abstract: The autosomal recessive human disorder ataxia-telangiectasia (A-T) was first described as a separate disease entity 40 years ago. It is a multisystem disease characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, radiosensitivity, predisposition to lymphoid malignancies and immunodeficiency, with defects in both cellular and humoral immunity. The pleiotropic nature of the clinical and cellular phenotype suggests that the gene product involved is important in maintaining stability of the genome but also plays a more general role in signal transduction. The chromosomal instability and radiosensitivity so characteristic of this disease appear to be related to defective activation of cell cycle checkpoints. Greater insight into the nature of the defect in A-T has been provided by the recent identification, by positional cloning, of the responsible gene, ATM. The ATM gene is related to a family of genes involved in cellular responses to DNA damage and/or cell cycle control. These genes encode large proteins containing a phosphatidylinositol 3-kinase domain, some of which have protein kinase activity. The mutations causing A-T completely inactivate or eliminate the ATM protein. This protein has been detected and localized to different subcellular compartments.

ATAXIA-TELANGIECTASIA AND THE NIJMEGEN BREAKAGE SYNDROME: Related Disorders But Genes Apart

Abstract: ▪ Abstract Gene mutations provide valuable clues to cellular metabolism. In humans such insights come mainly from genetic disorders. Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are two distinct, but closely related, single gene disorders that highlight a complex junction of several signal transduction pathways. These pathways appear to control defense mechanisms against specific types of damage to cellular macromolecules, and probably regulate the processing of certain types of DNA damage or normal intermediates of DNA metabolism. A-T is characterized primarily by cerebellar degeneration, immunodeficiency, genome instability, clinical radiosensitivity, and cancer predisposition. NBS shares all these features except cerebellar deterioration. The cellular phenotypes of A-T and NBS are almost indistinguishable, however, and include chromosomal instability, radiosensitivity, and defects in cell cycle checkpoints normally induced by ionizing radiation. The recent identification of the gene...

Interaction between ATM protein and c-Abl in response to DNA damage

Abstract: The gene mutated in the autosomal recessive disorder ataxia telangiectasia (AT), designated ATM (for 'AT mutated'), is a member of a family of phosphatidylinositol-3-kinase-like enzymes that are involved in cell-cycle control, meiotic recombination, telomere length monitoring and DNA-damage response. Previous results have demonstrated that AT cells are hypersensitive to ionizing radiation and are defective at the G1/S checkpoint after radiation damage. Because cells lacking the protein tyrosine kinase c-Abl are also defective in radiation-induced G1 arrest, we investigated the possibility that ATM might interact with c-Abl in response to radiation damage. Here we show that ATM binds c-Abl constitutively in control cells but not in AT cells. Our results demonstrate that the SH3 domain of c-Abl interacts with a DPAPNPPHFP motif (residues 1,373-1,382) of ATM. The results also reveal that radiation-induction of c-Abl tyrosine kinase activity is diminished in AT cells. These findings indicate that ATM is involved in the activation of c-Abl by DNA damage and this interaction may in part mediate radiation-induced G1 arrest.

Recombinant ATM protein complements the cellular A-T phenotype

Abstract: Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability and radiation sensitivity. The cellular phenotype of A-T points to defects in signal transduction pathways involved in activation of cell cycle checkpoints by free radical damage, and other pathways that mediate the transmission of specific mitogenic stimuli. The product of the responsible gene, ATM, belongs to a family of large proteins that contribute to maintaining genome stability and cell cycle progression in various organisms. A recombinant vector that stably expresses a full-length ATM protein is a valuable tool for its functional analysis. We constructed and cloned a recombinant, full-length open reading frame of ATM using a combination of vectors and hosts that overcame an inherent instability of this sequence. Recombinant ATM was stably expressed in insect cells using a baculovirus vector, albeit at a low level, and in human A-T cells using an episomal expression vector. An amino-terminal FLAG epitope added to the protein allowed highly specific detection of the recombinant molecule by immunoblotting, immunoprecipitation and immunostaining, and its isolation using immunoaffinity. Similar to endogenous ATM, the recombinant protein is located mainly in the nucleus, with low levels in the cytoplasm. Ectopic expression of ATM in A-T cells restored normal sensitivity to ionizing radiation and the radiomimetic drug neocarzinostatin, and a normal pattern of post-irradiation DNA synthesis, which represents an S-phase checkpoint. These observations indicate that the recombinant, epitope-tagged protein is functional. Introduction into this molecule of a known A-T missense mutation, Glu2904Gly, resulted in apparent instability of the protein and inability to complement the A-T phenotype. These findings indicate that the physiological defects characteristic of A-T cells result from the absence of the ATM protein, and that this deficiency can be corrected by ectopic expression of this protein.

The ataxia-telangiectasia gene product, a constitutively expressed nuclear protein that is not up-regulated following genome damage

Abstract: The product of the ataxia-telangiectasia gene (ATM) was identified by using an antiserum developed to a peptide corresponding to the deduced amino acid sequence. The ATM protein is a single, high-molecular weight protein predominantly confined to the nucleus of human fibroblasts, but is present in both nuclear and microsomal fractions from human lymphoblast cells and peripheral blood lymphocytes. ATM protein levels and localization remain constant throughout all stages of the cell cycle. Truncated ATM protein was not detected in lymphoblasts from ataxia-telangiectasia patients homozygous for mutations leading to premature protein termination. Exposure of normal human cells to γ-irradiation and the radiomimetic drug neocarzinostatin had no effect on ATM protein levels, in contrast to a noted rise in p53 levels over the same time interval. These findings are consistent with a role for the ATM protein in ensuring the fidelity of DNA repair and cell cycle regulation following genome damage.

Ataxia-telangiectasia: is ATM a sensor of oxidative damage and stress?

Abstract: Ataxia-telangiectasia (A-T) is a pleiotropic recessive disorder characterized by cerebellar ataxia, immunodeficiency, specific developmental defects, profound predisposition to cancer and acute radiosensitivity. Functional inactivation of a single gene product, ATM, accounts for this compound phenotype. We suggest that ATM acts as a sensor of reactive oxygen species and/or oxidative damage of cellular macromolecules, including DNA. In turn, ATM induces signalling through multiple pathways, thereby coordinating acute phase stress responses with cell cycle checkpoint control and repair of oxidative damage. Absence of ATM is proposed to limit the repair of insidious oxidative damage that can occur under normal physiological conditions, ultimately leading to apoptosis of particularly sensitive cells, such as neurons and thymocytes.

Ataxia-Telangiectasia Locus: Sequence Analysis of 184 kb of Human Genomic DNA Containing the Entire ATM Gene

Abstract: Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity, and cancer predisposition. The genomic organization of the A-T gene, designated ATM, was established recently. To date, more than 100 A-T-associated mutations have been reported in the ATM gene that do not support the existence of one or several mutational hotspots. To allow genotype/phenotype correlations it will be important to find additional ATM mutations. The nature and location of the mutations will also provide insights into the molecular processes that underly the disease. To facilitate the search for ATM mutations and to establish the basis for the identification of transcriptional regulatory elements, we have sequenced and report here 184,490 bp of genomic sequence from the human 11q22-23 chromosomal region containing the entire ATM gene, spanning 146 kb, and 10 kb of the 5'-region of an adjacent gene named E14/NPAT. The latter shares a bidirectional promoter with ATM and is transcribed in the opposite direction. The entire region is transcribed to approximately 85% and translated to 5%. Genome-wide repeats were found to constitute 37.2%, with LINE (17.1%) and Alu (14.6%) being the main repetitive elements. The high representation of LINE repeats is attributable to the presence of three full-length LINE-1s, inserted in the same orientation in introns 18 and 63 as well as downstream of the ATM gene. Homology searches suggest that ATM exon 2 could have derived from a mammalian interspersed repeat (MIR). Promoter recognition algorithms identified divergent promoter elements within the CpG island, which lies between the ATM and E14/NPAT genes, and provide evidence for a putative second ATM promoter located within intron 3, immediately upstream of the first coding exon. The low G+C level (38.1%) of the ATM locus is reflected in a strongly biased codon and amino acid usage of the gene.

Loss of heterozygosity at 11q23.1 in breast carcinomas: Indication for involvement of a gene distal and close to ATM

Abstract: Previous reports have suggested that heterozygotes for ataxia-telangiectasia (A-T) have an increased risk of cancer, in particular breast cancer. The ATM gene, responsible for A-T, was recently cloned. Loss of heterozygosity (LOH) in the chromosome band 11q23, where the ATM gene is located, has been reported in several types of tumours including breast carcinomas. Whether the ATM gene is the target, and the sole target, for the LOH seen in this region is not yet known. In this study, 169 primary breast carcinomas and 10 metastases were examined for allelic imbalance (AI) using 10 microsatellite markers mapping to 11q23.1. Nine of the markers reside within a 10 Mb region surrounding the ATM gene, whereas the tenth locus, APOC-3, is located more than 12 Mb telomeric from this region. The highest frequencies of alteration were found for APOC-3 (45%), and for two markers located approximately 200 and 900 kb telomeric from ATM, D11S1294 (44%) and D11S1818 (44%). The marker located within the ATM gene, D11S2179, was altered in 37% of the informative tumours. The present deletion map indicates that three distinct regions at 11q23.1 may be involved in breast cancer development; one between the markers D11S1294 and D11S1818, a second close to APOC-3, and a third that is possibly the ATM-gene itself.

Ataxia-telangiectasia: structural diversity of untranslated sequences suggests complex post-transcriptional regulation of ATM gene expression

Abstract: Mutations in the ATM gene are responsible for the multisystem disorder ataxia-telangiectasia, characterized by neurodegeneration, immune deficiency and cancer predisposition. While no alternative splicing was identified within the coding region, the first four exons of the ATM gene, which fall within the 54 untranslated region (UTR), undergo extensive alternative splicing. We identified 12 different 54 UTRs that show considerable diversity in length and sequence contents. These mRNA leaders, which range from 150 to 884 nucleotides (nt), are expected to form variable secondary structures and contain different numbers of AUG codons. The longest 54 UTR contains a total of 18 AUGs upstream of the translation start site. The 34 UTR of 3590 nt is contained within a single 34 exon. Alternative polyadenylation results in 34 UTRs of varying lengths. These structural features suggest that ATM expression might be subject to complex posttranscriptional regulation, enabling rapid modulation of ATM protein level in response to environmental stimuli or alterations in cellular physiological states.