RESUMO
The Type VI secretion system (T6SS) mediates toxin delivery into both eukaryotic and prokaryotic cells. It is composed of a cytoplasmic structure resembling the tail of contractile bacteriophages anchored to the cell envelope through a membrane complex composed of the TssL and TssM inner membrane proteins and of the TssJ outer membrane lipoprotein. The C-terminal domain of TssM is required for its interaction with TssJ, and for the function of the T6SS. In Citrobacter rodentium, the tssM1 gene does not encode the C-terminal domain. However, the stop codon is preceded by a run of 11 consecutive adenosines. In this study, we demonstrate that this poly-A tract is a transcriptional slippery site that induces the incorporation of additional adenosines, leading to frameshifting, and hence the production of two TssM1 variants, including a full-length canonical protein. We show that both forms of TssM1, and the ratio between these two forms, are required for the function of the T6SS in C. rodentium. Finally, we demonstrate that the tssM gene associated with the Yersinia pseudotuberculosis T6SS-3 gene cluster is also subjected to transcriptional frameshifting.
Assuntos
Sistemas de Secreção Bacterianos/genética , Citrobacter rodentium/genética , Citrobacter rodentium/metabolismo , Códon sem Sentido , Mutação da Fase de Leitura/fisiologia , Proteínas de Membrana/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/metabolismo , Sequência de Bases , Dados de Sequência Molecular , Organismos Geneticamente Modificados , Isoformas de Proteínas/genética , Análise de Sequência de DNA , Supressão GenéticaRESUMO
BACKGROUND: Mutations in the genes encoding for GHRH receptor (GHRHR) and GH (GH1) are the most common cause of familial isolated GH deficiency (IGHD). GHRHR mutations are often associated with anterior pituitary hypoplasia (APH), but this has been reported almost exclusively in children older than 8 yr. We analyzed the GHRHR and measured pituitary size in a consanguineous family with the father and three of the five siblings with IGHD. OBJECTIVE: The aim of the study was to find the mutated gene in a family with severe IGHD. METHODS: We sequenced the whole GHRHR coding regions and the intron-exon boundaries from peripheral DNA of the index patient. After identifying the novel mutation, we sequenced the region of interest in the other members of the family. We measured the anterior pituitary volume from magnetic resonance imaging (MRI). RESULTS: The father and the three affected children were homozygous for a new frame-shift mutation in the coding sequence of exon 4 (corresponding to the extracellular domain of the receptor) (c.340delG) that places the downstream sequence out of frame [corrected]. The mother and two unaffected siblings were heterozygous for the mutation. Two of the affected children had MRI evidence of APH before reaching 6 yr of age. CONCLUSIONS: We describe a new mutation in the GHRHR in a family with IGHD. The presence of frank APH before age 6 yr shows that MRI-evident reduced pituitary size can be present in GHRHR mutations even in children younger than 8 yr of age.
Assuntos
Mutação da Fase de Leitura/genética , Mutação da Fase de Leitura/fisiologia , Hormônio do Crescimento Humano/deficiência , Doenças da Hipófise/patologia , Adeno-Hipófise/patologia , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/fisiologia , Receptores de Hormônios Reguladores de Hormônio Hipofisário/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário/fisiologia , Adolescente , Estatura/fisiologia , Criança , Pré-Escolar , DNA/genética , Éxons/genética , Feminino , Transtornos do Crescimento/etiologia , Terapia de Reposição Hormonal , Hormônio do Crescimento Humano/uso terapêutico , Humanos , Íntrons/genética , Imageamento por Ressonância Magnética , Linhagem , Proteínas Recombinantes/uso terapêuticoRESUMO
Alexander disease is a primary genetic disorder of astrocyte caused by dominant mutations in the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP). While most of the disease-causing mutations described to date have been found in the conserved α-helical rod domain, some mutations are found in the C-terminal non-α-helical tail domain. Here, we compare five different mutations (N386I, S393I, S398F, S398Y and D417M14X) located in the C-terminal domain of GFAP on filament assembly properties in vitro and in transiently transfected cultured cells. All the mutations disrupted in vitro filament assembly. The mutations also affected the solubility and promoted filament aggregation of GFAP in transiently transfected MCF7, SW13 and U343MG cells. This correlated with the activation of the p38 stress-activated protein kinase and an increased association with the small heat shock protein (sHSP) chaperone, αB-crystallin. Of the mutants studied, D417M14X GFAP caused the most significant effects both upon filament assembly in vitro and in transiently transfected cells. This mutant also caused extensive filament aggregation coinciding with the sequestration of αB-crystallin and HSP27 as well as inhibition of the proteosome and activation of p38 kinase. Associated with these changes were an activation of caspase 3 and a significant decrease in astrocyte viability. We conclude that some mutations in the C-terminus of GFAP correlate with caspase 3 cleavage and the loss of cell viability, suggesting that these could be contributory factors in the development of Alexander disease.
Assuntos
Doença de Alexander/genética , Caspase 3/metabolismo , Sobrevivência Celular/genética , Proteína Glial Fibrilar Ácida/genética , Filamentos Intermediários/metabolismo , Mutação/fisiologia , Doença de Alexander/etiologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Astrocitoma/patologia , Linhagem Celular Tumoral , Centrifugação , Ciclina D1/metabolismo , Epitopos/imunologia , Mutação da Fase de Leitura/fisiologia , Proteína Glial Fibrilar Ácida/imunologia , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico , Humanos , Filamentos Intermediários/patologia , Filamentos Intermediários/ultraestrutura , Microscopia Eletrônica de Transmissão , Chaperonas Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto/fisiologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade , Transfecção , Ubiquitina/metabolismo , Vimentina/metabolismo , Cadeia B de alfa-Cristalina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
INTRODUCTION: Germline mutations in the NF2 gene are responsible for 80 p.cent of neurofibromatosis type 2 typical cases. Mutations are mainly truncating mutations or deletions, missense mutations having been reported in few cases. An important phenotypic variability is observed among gene carriers. To assess whether the phenotypic variability of neurofibromatosis 2 could be linked to genotype, clinical data of 154 patients whose NF2 germline alteration had been identified in our laboratory have been collected. METHODS: A retrospective questionnaire was sent to the physicians in charge of these patients. Statistical analyses regarding genotypic and phenotypic data were performed by comparisons of average values and correlation tests. RESULTS: In French patients, type of mutation was correlated neither with patients' sex, nor with disease occurrence mode (de novo or inherited mutation). Disease associated with missense mutations occurred later, with a less severe symptomatology. Patients with nonsense or frameshift mutations were more frequently affected with meningiomas and spinal tumours, in addition to VIII nerve schwannomas, an observation that underlies the genetic determination of the number and type of NF2-related tumours. CONCLUSION: Results from the literature as well as from our study tend to show that only few correlations exist between genotype and phenotype in the NF2 disease. It also recognizes that missense mutations have a lower level of evolution, severity and mortality risk. Nonsense and frameshift mutations seem to be associated with a higher number of meningiomas and spinal tumours. Therefore, NF2 gene screening keeps its indications in both typical and moderate forms of the disease. Mutations are responsible of 80 p.cent of typical forms; in moderate forms, identification of a missense mutation seems linked to a lower disease evolution. In any case, assessment and supervision should be identical. Finally, in a small number of cases, the NF2 gene appears to be implicated in clinical forms different from those defined by NIH and it might be of interest to enlarge the clinical features suggestive of the disease.
Assuntos
Genes da Neurofibromatose 2/fisiologia , Heterozigoto , Neurofibromatose 2/genética , Neurofibromatose 2/fisiopatologia , Adulto , Códon sem Sentido/genética , Códon sem Sentido/fisiologia , Feminino , Mutação da Fase de Leitura/genética , Mutação da Fase de Leitura/fisiologia , França/epidemiologia , Genótipo , Humanos , Masculino , Meningioma/epidemiologia , Meningioma/etiologia , Meningioma/genética , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/fisiologia , Neurofibromatose 2/epidemiologia , Fenótipo , Médicos , Estudos Retrospectivos , Medição de Risco , Neoplasias da Coluna Vertebral/epidemiologia , Neoplasias da Coluna Vertebral/etiologia , Neoplasias da Coluna Vertebral/genética , Inquéritos e QuestionáriosRESUMO
DNA polymerase beta (Pol beta) is important for the base excision repair (BER) pathway. Overexpression of Pol beta is frequently found in cancer cells and is thought to be associated with tumorigenesis. In this study, we examined BER fidelity in extracts derived from a human lymphoblastoid cell line that over expresses Pol beta compared to normal control cells. Using an in vitro mutagenesis assay, we found an increased rate of frameshift mutations arising during DNA repair in whole-cell extracts derived from the Pol beta-overexpressing cells. We demonstrate that the addition of excess Pol beta to a control cell extract enhances the mutagenic potential of the extract. Furthermore, using cell extracts and purified Pol beta, we demonstrate that the mechanism of frameshift formation involves slippage of Pol beta during the one-nucleotide gap-filling step of BER and that this slippage is fixed by strand-displacement synthesis stimulated by an excess of Pol beta.
Assuntos
DNA Polimerase beta/farmacologia , Reparo do DNA/fisiologia , Mutação da Fase de Leitura/efeitos dos fármacos , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , DNA Polimerase beta/metabolismo , Replicação do DNA/fisiologia , Escherichia coli , Mutação da Fase de Leitura/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Dados de Sequência Molecular , OligonucleotídeosRESUMO
We examined mismatch repair (MMR)-defective diploid strains of budding yeast grown for approximately 160 generations to determine whether decreases in spore viability due to the uncovering of recessive lethal mutations correlated with an increase in gross chromosomal rearrangements (GCRs). No GCRs were detected despite dramatic decreases in spore viability, suggesting that frameshift and/or other unrepaired DNA replication lesions play a greater role than chromosomal instability in decreasing viability in MMR-defective strains.
Assuntos
Pareamento Incorreto de Bases/genética , Aberrações Cromossômicas , Proteínas Fúngicas/genética , Mutação/fisiologia , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal , Sobrevivência Celular/genética , Mutação da Fase de Leitura/fisiologia , Rearranjo Gênico/fisiologia , Genes Letais/fisiologia , Genes Recessivos/fisiologia , Modelos Biológicos , Proteína 1 Homóloga a MutL , Hibridização de Ácido Nucleico/métodos , Probabilidade , Proteínas de Saccharomyces cerevisiaeRESUMO
BACKGROUND & AIMS: The NOD2 variants R702W, G908R, and L1007fsinsC are strongly associated with Crohn's disease (CD) in both European and American populations, but whether this susceptibility extends to all ethnic groups remains unknown. Except for the L1007fsinsC mutation, which produces a truncated NOD2 protein, the functional activity of the major CD-associated variants G908R and R702W is unknown. METHODS: Individuals were genotyped for R702W, G908R, and L1007fsinsC. The ability of G908R, R702W, and L1007fsinsC variants in the presence and absence of P268S to confer responsiveness to lipopolysaccharide (LPS) and peptidoglycan (PGN) was determined in HEK293T kidney cells. RESULTS: G908R and L1007fsinsC, but not R702W, were associated with disease susceptibility in Ashkenazi Jews. Ashkenazi Jews with CD had significantly higher allele frequency carriage of G908R and lower carriage of R702W compared with non-Jewish whites with CD. Functional studies revealed that the G908R, R702W, and L1007fsinsC variants in the presence and absence of P268S are defective in their ability to respond to bacterial LPS and PGN, whereas P268S alone exhibited wild-type activity. CONCLUSIONS: R702W is not associated with susceptibility to CD in Ashkenazi Jews. The G908R, R702W, and L1007fsinsC variants share a common signaling defect in response to bacterial components, providing evidence for a unifying molecular mechanism whereby NOD2 mutations contribute to disease susceptibility.
Assuntos
Proteínas de Transporte/genética , Doença de Crohn/genética , Doença de Crohn/fisiopatologia , Variação Genética/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/farmacologia , Peptidoglicano/farmacologia , Transdução de Sinais/efeitos dos fármacos , Alelos , Estudos de Casos e Controles , Linhagem Celular , Mutação da Fase de Leitura/fisiologia , Predisposição Genética para Doença/genética , Genótipo , Heterozigoto , Humanos , Judeus , Proteína Adaptadora de Sinalização NOD2RESUMO
Unraveling the molecular basis of inherited disorders of epithelial fragility has led to understanding of the complex structure and function of keratin intermediate filaments. Keratins are organized as a central alpha-helical rod domain flanked by nonhelical, variable end domains. Pathogenic mutations in 19 different keratin genes have been identified in sequences corresponding to conserved regions at the beginning and end of the rod. These areas have been recognized as zones of overlap between aligned keratin proteins and are thought to be crucial for proper assembly of keratin intermediate filaments. Consequently, all keratin disorders of skin, hair, nail, and mucous membranes caused by mutations in rod domain sequences are characterized by perinuclear clumping of fragmented keratin intermediate filaments, thus compromising mechanical strength and cell integrity. We report here the first mutation in a keratin gene (KRT1) that affects the variable tail domain (V2) and results in a profoundly different abnormality of the cytoskeletal architecture leading to a severe form of epidermal hyperkeratosis known as ichthyosis hystrix Curth-Macklin. Structural analyses disclosed a failure in keratin intermediate filament bundling, retraction of the cytoskeleton from the nucleus, and failed translocation of loricrin to the desmosomal plaques. These data provide the first in vivo evidence for the crucial role of a keratin tail domain in supramolecular keratin intermediate filament organization and barrier formation.
Assuntos
Mutação da Fase de Leitura/fisiologia , Ictiose/genética , Ictiose/fisiopatologia , Queratinas/genética , Queratinas/fisiologia , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Citoplasma/metabolismo , Heterozigoto , Humanos , Ictiose/patologia , Filamentos Intermediários/fisiologia , Filamentos Intermediários/ultraestrutura , Ceratodermia Palmar e Plantar/genética , Ceratodermia Palmar e Plantar/patologia , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Dados de Sequência Molecular , Linhagem , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína/genética , Distribuição TecidualRESUMO
We report a novel mutation in the XK gene (XK) in a Japanese patient with McLeod syndrome. A 50-year-old man showed progressive muscular atrophy, choreic movement, elevated level of serum creatinine kinase, and acanthocytosis. The expression level of all the Kell antigens in erythrocyte was decreased and molecular analysis revealed a single-base (T) deletion at the nucleotide position 1095 in XK. This deletion caused a frameshift in translation, leading to a premature stop codon at the amino acid position 408. We conclude this single-base deletion causes defective Kx protein, which is responsible for the McLeod phenotype in this patient.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Mutação da Fase de Leitura/fisiologia , Doenças Neuromusculares/genética , Cromossomo X/genética , Antígenos de Superfície/biossíntese , Antígenos de Superfície/genética , Proteínas Sanguíneas/biossíntese , Proteínas Sanguíneas/genética , Encéfalo/diagnóstico por imagem , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , DNA/análise , DNA/genética , Citometria de Fluxo , Mutação da Fase de Leitura/genética , Ligação Genética/genética , Ligação Genética/fisiologia , Sistema Hematopoético/fisiopatologia , Humanos , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Doenças Neuromusculares/diagnóstico por imagem , Doenças Neuromusculares/fisiopatologia , Síndrome , Tomografia Computadorizada por Raios XRESUMO
Mice homozygous for the recessive mutation oscillator (Glra1(spd-ot)) suffer from a complex motor disorder leading to death within three weeks after birth. Symptoms of this disorder mimic poisoning by strychnine, the antagonist of the inhibitory glycine receptor. The syndrome has previously been correlated to a 7 base pair microdeletion within the Glra1 gene (chromosome 11) encoding the alpha1-subunit of the adult glycine receptor isoform. As shown by [3H]strychnine binding and western blot analysis employing subunit-specific antibodies, spinal cord of homozygous oscillator mice was totally devoid of alpha1-polypeptide, characterizing the Glra1(spd-ot) gene as a functional null allele of Glra1. Moreover, tissue levels of the postsynaptic anchoring protein gephyrin were drastically reduced in the Glra1(spd-ot)/Glra1(spd-ot) genotype. In contrast, immunoanalysis revealed a persisting low-level expression of non-alpha1 glycine receptor polypeptides. Spinal glycine receptor content was also significantly reduced in the +/Glra1(spd-ot) genotype. This reduction coincided with increased acoustic startle responses in heterozygous animals consistent with haplotype insufficiency of glycine receptor function. Lethality of the murine null allele Glra1(spd-ot) contrasts with the situation in the human, where homozygosity for a GLRA1 null allele produces the phenotype of the non-lethal disorder hyperekplexia (startle disease; stiff baby syndrome). This suggests a disparate regulation of glycine receptor subunit genes and/or diverse compensatory pathways in mice and humans.
Assuntos
Sistema Nervoso Central/metabolismo , Mutação da Fase de Leitura/fisiologia , Receptores de Glicina/genética , Estimulação Acústica , Animais , Animais Recém-Nascidos , Western Blotting , Feminino , Masculino , Membranas/metabolismo , Camundongos , Camundongos Mutantes Neurológicos , Fenótipo , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Receptores de Glicina/biossíntese , Reflexo de Sobressalto/fisiologia , Estricnina/metabolismoRESUMO
YY1 is a C2H2-type zinc finger transcription factor that is a member of the human GLl-Kruppel family of proteins. YY1 represses transcription when bound upstream of transcription initiation sites. The repression can be relieved by adenovirus E1A and activation of target genes occurs. We have mapped the repression domain of YY1 to the C-terminal region, overlapping its DNA binding domain. We have also identified an activation domain within the first 69 amino acids of YY1. The YY1 C-terminal region is involved in physical interactions with E1A and is functionally necessary for YY1 to respond to E1A. This suggests that relief of YY1 repression by E1A involves YY1-E1A physical interactions. Although not involved in interactions with E1A, the N-terminal activation domain is also necessary for YY1 to respond to E1A. Presumably, under repressing conditions, the activation domain is masked by the conformation of YY1, but is released upon binding of E1A and is required to subsequently activate transcription. Consistent with this hypothesis, an ATF-2-YY1 chimeric protein containing the activation domain of ATF-2 and the C-terminal two-thirds of YY1 is still a potent repressor. Unlike the mutant YY1 lacking its own N-terminal activation domain, the chimeric protein is fully responsive to E1A.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Fator 2 Ativador da Transcrição , Proteínas E1A de Adenovirus/genética , Sequência de Aminoácidos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Fatores de Ligação de DNA Eritroide Específicos , Mutação da Fase de Leitura/fisiologia , Células HeLa , Humanos , Conformação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência/fisiologia , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fator de Transcrição YY1RESUMO
A ribosomal frameshift at the gag-pro junction of mouse mammary tumor virus (MMTV) gives rise to the protein p30. The protein consists of two domains, the zinc-finger-containing nucleocapsid (NC) protein portion with 95 residues and a C-terminal extension comprising 154 residues. The C-terminal domain shows similarity in sequence with the enzyme dUTPase from other sources. In this paper, we demonstrate that p30 is a functional dUTPase. Overproduction of the NC protein in Escherichia coli, using the native frameshift sequence at the gag stop codon, caused a detectable expression of dUTPase ascribed to a low frequency of readthrough. By a 1-base insertion, eliminating the gag stop codon and fusing the gag and pro reading frames, a plasmid, pET-3d-NCDU, directing overexpression of p30, was constructed. The overproduced protein, purified by phosphocellulose chromatography, shows both zinc-binding and dUTPase activity. Analytical gel filtration and sequence homology to other dUTPases suggest a trimeric assembly of p30 subunits. MMTV thus possesses two different forms of the nucleocapsid protein, the ordinary NC protein and the p30, having the NC protein connected to a domain of dUTPase.
Assuntos
Capsídeo/metabolismo , Produtos do Gene gag/genética , Vírus do Tumor Mamário do Camundongo/química , Pirofosfatases/metabolismo , Proteínas do Core Viral/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Capsídeo/genética , Mutação da Fase de Leitura/fisiologia , Vírus do Tumor Mamário do Camundongo/enzimologia , Vírus do Tumor Mamário do Camundongo/genética , Dados de Sequência Molecular , Conformação Proteica , Pirofosfatases/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas do Core Viral/genética , Zinco/metabolismoRESUMO
The red clover necrotic mosaic virus (RCNMV) genome is split between two single-stranded RNA species termed RNA-1 and RNA-2. RNA-1 directs the synthesis of 88-kDa (p88), 57-kDa (p57), 37-kDa (p37), and 27-kDa (p27) polypeptides and RNA-2 a 35-kDa (p35) polypeptide in vitro. The coding order of the RNA-1 products was determined to be 5'-p27-p57-p37-3'. Antibodies to synthetic peptides representing the carboxyl terminal portions of p27 and p57 immunoprecipitated their respective polypeptides in addition to p88, suggesting that p88 is a fusion protein. A frameshift heptanucleotide sequence element has been identified in RCNMV RNA-1. In addition, a stable stem-loop secondary structure adjacent to the heptanucleotide sequence is predicted. Together, these sequence elements suggest that a ribosomal frameshifting event occurs which allows translational readthrough of the p27 open reading frame into the p57 open reading frame, generating the observed p88 product. An RNA-1 expression construct fusing the p57 and the CP open reading frame was engineered to investigate the ribosomal frameshifting event. CP antibodies immunoprecipitated a fusion protein of the predicted size containing the carboxyl portion of CP. Site-directed mutagenesis of the frameshift element indicates that in vitro, p88 can also be expressed alternatively by suppression of an amber termination codon. Based on these data, we propose that the putative RCNMV RNA polymerase is an 88-kDa polypeptide expressed by a ribosomal frameshifting mechanism similar to those utilized by retroviruses.