Autosomal SCID caused by a point mutation in the N-terminus of Jak3: mapping of the Jak3-receptor interaction domain.
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Signaling through the hematopoietic receptors requires activation of receptor-associated Janus (Jak) kinases. For example, Jak1 and Jak3 bind specifically to the IL-2 receptor beta (IL-2Rbeta) and common gamma (gammac) chains, respectively, and initiate biochemical signals critical in controlling immune responses. The region of Jak responsible for receptor interactions, however, is not well characterized. Here we describe a naturally occurring Jak3 mutation from a patient with autosomal severe combined immunodeficiency (SCID), where a single amino acid substitution, Y100C, in Janus homology domain 7 (JH7) prevents kinase-receptor interaction. This mutation also results in a loss of IL-2-induced signaling in a B-cell line derived from this patient. Using mutational analysis we have identified a region of Jak3, including portions of JH6 and JH7, that is sufficient for kinase-receptor contact and show that this segment interacts with the proline-rich Box1 region of the receptor. Furthermore, a Jak3-Jak1 chimera containing only the JH6 and JH7 domains of Jak3 interacts with gammac and can reconstitute IL-2-dependent responses, including receptor phosphorylation and activation of signal transducer and activator of transcription (STAT) 5b. Our results suggest that the N-terminus of Jak kinases is critical for receptor binding, and is therefore likely to determine specificity of Jak kinase-receptor interactions. (+info)
Mutual activation of Ets-1 and AML1 DNA binding by direct interaction of their autoinhibitory domains.
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The transcription factors Ets-1 and AML1 (the alphaBl subunit of PEBP2/CBF) play critical roles in hematopoiesis and leukemogenesis, and cooperate in the transactivation of the T cell receptor (TCR) beta chain enhancer. The DNA binding capacity of both factors is blocked intramolecularly but can be activated by the removal of negative regulatory domains. These include the exon VII domain for Ets-1 and the negative regulatory domain for DNA binding (NRDB) for alphaB1. Here we report that the direct interaction between the two factors leads to a reciprocal stimulation of their DNA binding activity and activation of their transactivation function. Detailed mapping revealed two independent contact points involving the exon VII and NRDB regions as well as the two DNA binding domains. Using deletion variants and dominant interfering mutants, we demonstrate that the interaction between exon VII and NRDB is necessary and sufficient for cooperative DNA binding. The exon VII and NRDB motifs are highly conserved in evolution yet deleted in natural variants, suggesting that the mechanism described is of biological relevance. The mutual activation of DNA binding of Ets and AML1 through the intermolecular interaction of autoinhibitory domains may represent a novel principle for the regulation of transcription factor function. (+info)
The repressor which binds the -75 GATA motif of the GPB promoter contains Ku70 as the DNA binding subunit.
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Glycophorin B (GPB) is an abundant cell surface glycoprotein which is only expressed in human erythroid cells. Previous functional analysis demonstrated that the repression of the GPB promoter is determined by the binding of a ubiquitous factor which recognizes a GATA motif centered at position -75. In erythroid cells this ubiquitous factor is displaced by the binding of the erythroid-specific factor hGATA1. Here, we have identified the Ku70 protein as a candidate GPB repressor DNA binding subunit through the screening of a human HeLa expression library using the -75 GATA sequence as bait (one-hybrid method). Electrophoretic mobility shift assays demonstrated that the ubiquitous factor that binds the -75 GATA sequence was the Ku70-Ku80 (Ku) heterodimer. Co-transfection experiments demonstrated that overexpression of Ku70 in the K562 erythroleukeamic cell line resulted in transcriptional repression of the chloramphenicol acetyltransferase reporter gene when placed under the control of the wild-type GPB promoter. Conversely, no repression was observed when a mutation that abolished the binding of Ku was introduced in the GPB promoter construct. Altogether, these results indicate that Ku binds in vivo to the -75 WGATAR motif and is involved in negative regulation of the GPB promoter. These findings suggest that, besides its role in many functions, Ku is also involved in transcriptional regulation of erythroid genes. (+info)
Mitochondria in organismal aging and degeneration.
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Several lines of experimentation support the view that the genetic, biochemical and bioenergetic functions of somatic mitochondria deteriorate during normal aging. Deletion mutations of the mitochondrial genome accumulate exponentially with age in nerve and muscle tissue of humans and multiple other species. In muscle, a tissue that undergoes age-related fiber loss and atrophy in humans, there is an exponential rise in the number of cytochrome-oxidase-deficient fibers, which is first detectable in the fourth decile of age. Most biochemical studies of animal mitochondrial activity indicate a decline in electron transport activity with age, as well as decreased bioenergetic capacity with age, as measured by mitochondrial membrane potential. Mitochondrial mutations may be both the result of mitochondrial oxidative stress, and cells bearing pure populations of pathogenic mitochondrial mutations are sensitized to oxidant stress. Oxidant stress to mitochondria is known to induce the mitochondrial permeability transition, which has recently been implicated in the release of cytochrome c and the initiation of apoptosis. Thus several lines of evidence support a contribution of mitochondrial dysfunction to the phenotypic changes associated with aging. (+info)
Identification of mutations in the c-mpl gene in congenital amegakaryocytic thrombocytopenia.
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Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder expressed in infancy and characterized by isolated thrombocytopenia and megakaryocytopenia with no physical anomalies. Our previous hematological analysis indicated similarities between human CAMT and murine c-mpl (thrombopoietin receptor) deficiency. Because the c-mpl gene was considered as one of the candidate genes for this disorder, we analyzed the genomic sequence of the c-mpl gene of a 10-year-old Japanese girl with CAMT. We detected two heterozygous point mutations: a C-to-T transition at the cDNA nucleotide position 556 (Q186X) in exon 4 and a single nucleotide deletion of thymine at position 1,499 (1,499 delT) in exon 10. Both mutations were predicted to result in a prematurely terminated c-Mpl protein, which, if translated, lacks all intracellular domains essential for signal transduction. Each of the mutations was segregated from the patient's parents. Accordingly, the patient was a compound heterozygote for two mutations of the c-mpl gene, each derived from one of the parents. The present study suggests that at least a certain type of CAMT is caused by the c-mpl mutation, which disrupts the function of thrombopoietin receptor. (+info)
Analysis of the complete coding region of the CFTR gene in ten Algerian cystic fibrosis families.
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The spectrum of cystic fibrosis (CF) mutations in the North African population remains poorly known. In order to offer an effective diagnostic service and to determine accurate risk estimates, we decided to identify the CF mutations in 10 Algerian CF families. We carried out a chemical-clamp denaturing gradient gel electrophoresis analysis of the CFTR gene and automated direct DNA sequencing. We identified 5 mutations and we characterized 60% of the CF chromosomes. Taking advantage of the homogeneity of the sample, we report clinical features of homozygous CF patients. (+info)
Non-pseudogene-derived complex acid beta-glucosidase mutations causing mild type 1 and severe type 2 gaucher disease.
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Gaucher disease is an autosomal recessive inborn error of glycosphingolipid metabolism caused by the deficient activity of the lysosomal hydrolase, acid beta-glucosidase. Three phenotypically distinct subtypes result from different acid beta-glucosidase mutations encoding enzymes with absent or low activity. A severe neonatal type 2 variant who presented with collodion skin, ichthyosis, and a rapid neurodegenerative course had two novel acid beta-glucosidase alleles: a complex, maternally derived allele, E326K+L444P, and a paternally inherited nonsense mutation, E233X. Because the only other non-pseudogene-derived complex allele, D140H+E326K, also had the E326K lesion and was reported in a mild type 1 patient with a D140H+E326K/K157Q genotype, these complex alleles and their individual mutations were expressed and characterized. Because the E233X mutation expressed no activity and the K157Q allele had approximately 1% normal specific activity based on cross-reacting immunologic material (CRIM SA) in the baculovirus system, the residual activity in both patients was primarily from their complex alleles. In the type 1 patient, the D140H+E326K allele was neuroprotective, encoding an enzyme with a catalytic efficiency similar to that of the N370S enzyme. In contrast, the E326K+L444P allele did not have sufficient activity to protect against the neurologic manifestations and, in combination with the inactive E233X lesion, resulted in the severe neonatal type 2 variant. Thus, characterization of these novel genotypes with non-pseudogene-derived complex mutations provided the pathogenic basis for their diverse phenotypes. (+info)
Prognostic value of p53 genetic changes in colorectal cancer.
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PURPOSE: To explore whether there is a linkage between different mutations in the p53 gene in primary colorectal cancer and the risk of death from colorectal cancer in a large group of patients with long follow-up. We also compared a complementary DNA-based sequencing method and an immunohistochemical (IHC) method for detecting p53 protein overexpression in colorectal cancer. MATERIALS AND METHODS: The entire coding region of the p53 gene was sequenced in 191 frozen tumor samples collected from January 1988 to November 1992. RNA was extracted and synthesized to cDNA. p53 was amplified by the polymerase chain reaction, and the DO-7 monoclonal antibody was used in the IHC assessments. RESULTS: Mutations were detected in 99 samples (52%) from 189 patients. There was a significant relationship between the p53 mutational status and the cancer-specific survival time, with shorter survival time for patients who had p53 mutations than for those who did not (P = .01, log-rank test). Mutations outside the evolutionarily conserved regions were associated with the worst prognosis. Multivariate analysis showed that the presence of p53 mutations was an independent prognostic factor (relative hazard, 1.7, P = .03). There was no significant relationship between overexpression of p53 protein, as determined by IHC analysis, and cancer-specific survival. CONCLUSION: Mutational analyses of the p53 gene, using cDNA sequencing in colorectal cancer, provide useful prognostic information. In addition, cDNA sequencing gives better prognostic information than IHC assessment of p53 protein overexpression. (+info)