RESUMO
We report the mapping of a second myotonic dystrophy locus, myotonic dystrophy type 2 (DM2). Myotonic dystrophy (DM) is a multi-system disease and the most common form of muscular dystrophy in adults. In 1992, DM was shown to be caused by an expanded CTG repeat in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK) on chromosome 19 (refs 2-6). Although several theories have been put forth to explain how the CTG expansion causes the broad spectrum of clinical features associated with DM, it is not understood how this mutation, which does not alter the protein-coding region of a gene, causes an affect at the cellular level. We have identified a five-generation family (MN1) with a genetically distinct form of myotonic dystrophy. Affected members exhibit remarkable clinical similarity to DM (myotonia, proximal and distal limb weakness, frontal balding, cataracts and cardiac arrhythmias) but do not have the chromosome-19 D CTG expansion. We have mapped the disease locus (DM2) of the MN1 family to a 10-cM region of chromosome 3q. Understanding the common molecular features of two different forms of the disease should shed light on the mechanisms responsible for the broad constellation of seemingly unrelated clinical features present in both diseases.
Assuntos
Cromossomos Humanos Par 3 , Distrofia Miotônica/genética , Adulto , Mapeamento Cromossômico , Ligação Genética , Haplótipos , Humanos , LinhagemRESUMO
Myotonic dystrophy (DM) is the only disease reported to be caused by a CTG expansion. We now report that a non-coding CTG expansion causes a novel form of spinocerebellar ataxia (SCA8). This expansion, located on chromosome 13q21, was isolated directly from the genomic DNA of an ataxia patient by RAPID cloning. SCA8 patients have expansions similar in size (107-127 CTG repeats) to those found among adult-onset DM patients. SCA8 is the first example of a dominant SCA not caused by a CAG expansion translated as a polyglutamine tract.
Assuntos
Degenerações Espinocerebelares/genética , Repetições de Trinucleotídeos , Regiões não Traduzidas , Alelos , Feminino , Genes Dominantes , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase , Técnica de Amplificação ao Acaso de DNA Polimórfico , Transcrição GênicaRESUMO
Trinucleotide repeat expansions have been shown to cause a number of neurodegenerative diseases. A hallmark of most of these diseases is the presence of anticipation, a decrease in the age at onset in consecutive generations due to the tendency of the unstable trinucleotide repeat to lengthen when passed from one generation to the next. The involvement of trinucleotide repeat expansions in a number of other diseases--including familial spastic paraplegia, schizophrenia, bipolar affective disorder and spinocerebellar ataxia type 7 (SCA7; ref. 10)--is suggested both by the presence of anticipation and by repeat expansion detection (RED) analysis of genomic DNA samples. The involvement of trinucleotide expansions in these diseases, however, can be conclusively confirmed only by the isolation of the expansions present in these populations and detailed analysis to assess each expansion as a possible pathogenic mutation. We describe a novel procedure for quick isolation of expanded trinucleotide repeats and the corresponding flanking nucleotide sequence directly from small amounts of genomic DNA by a process of Repeat Analysis, Pooled Isolation and Detection of individual clones containing expanded trinucleotide repeats (RAPID cloning). We have used this technique to clone the pathogenic SCA7 CAG expansion from an archived DNA sample of an individual affected with ataxia and retinal degeneration.
Assuntos
Ataxia/genética , Clonagem Molecular/métodos , Distrofia Miotônica/genética , Degeneração Retiniana/genética , Repetições de Trinucleotídeos , Ataxia/patologia , Ataxina-7 , Sequência de Bases , Cromossomos Humanos Par 19 , DNA Complementar , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Distrofia Miotônica/patologia , Proteínas do Tecido Nervoso/genética , Linhagem , Reação em Cadeia da Polimerase , Degeneração Retiniana/patologiaRESUMO
Raine syndrome is an autosomal recessive disorder caused by mutations in the FAM20C gene. FAM20C codes for the human homolog of DMP4, a dentin matrix protein highly expressed in odontoblasts and moderately in bone. DMP4 is probably playing a role in the mineralization process. Since the first case reported in 1989 by Raine et al. 21 cases have been published delineating a phenotype which associates dysmorphic features, cerebral calcifications, choanal atresia or stenosis and thoracic/pulmonary hypoplasia. Kan and Kozlowski suggested the name of Raine syndrome to describe this new lethal osteosclerotic bone dysplasia. All the cases described were lethal during the neonatal period except for the last two reported patients aged 8 and 11 years who presented severe mental retardation. Here we describe two sisters, with an attenuated phenotype of Raine syndrome, who present an unexpectedly normal psychomotor development at ages 4 and 1, respectively. Identification of a homozygous mutation in the FAM20C gene confirmed the Raine syndrome diagnosis, thus contributing to the expansion of the Raine syndrome phenotype. This case report also prompted us to revisit the FAM20 gene classification and allowed us to highlight the ancestral status of Fam20C.
Assuntos
Anormalidades Múltiplas/genética , Fissura Palatina/genética , Exoftalmia/genética , Proteínas da Matriz Extracelular/genética , Microcefalia/genética , Mutação , Osteosclerose/genética , Anormalidades Múltiplas/diagnóstico , Sequência de Aminoácidos , Sequência de Bases , Osso e Ossos/patologia , Caseína Quinase I , Criança , Pré-Escolar , Atresia das Cóanas/genética , Atresia das Cóanas/metabolismo , Fissura Palatina/diagnóstico , Exoftalmia/diagnóstico , Feminino , Humanos , Masculino , Microcefalia/diagnóstico , Dados de Sequência Molecular , Osteosclerose/diagnóstico , FenótipoRESUMO
We report a novel form of modulation of T-type calcium currents carried out by the neuronal actin-binding protein (ABP) Kelch-like 1 (KLHL1). KLHL1 is a constitutive neuronal ABP localized to the soma and dendritic arbors; its genetic elimination in Purkinje neurons leads to dendritic atrophy and motor insufficiency. KLHL1 participates in neurite outgrowth and upregulates voltage-gated P/Q-type calcium channel function; here we investigated KLHL1's role as a modulator of low-voltage-gated calcium channels and determined the molecular mechanism of this modulation with electrophysiology and biochemistry. Coexpression of KLHL1 with Ca(V)3.1 or Ca(V)3.2 (alpha(1G) or alpha(1H) subunits) caused increases in T-type current density (35%) and calcium influx (75-83%) when carried out by alpha(1H) but not by alpha(1G). The association between KLHL1 and alpha(1H) was determined by immunoprecipitation and immunolocalization in brain membrane fractions and in vitro in HEK-293 cells. Noise analysis showed that neither alpha(1H) single-channel conductance nor open probability was altered by KLHL1, yet a significant increase in channel number was detected and further corroborated by Western blot analysis. KLHL1 also induced an increase in alpha(1H) current deactivation time (tau(deactivation)). Interestingly, the majority of KLHL1's effects were eliminated when the actin-binding motif (kelch) was removed, with the exception of the calcium influx increase during action potentials, indicating that KLHL1 interacts with alpha(1H) and actin and selectively regulates alpha(1H) function by increasing the number of alpha(1H) channels. This constitutes a novel regulatory mechanism of T-type calcium currents and supports the role of KLHL1 in the modulation of cellular excitability.
Assuntos
Actinas/metabolismo , Canais de Cálcio Tipo T/metabolismo , Cálcio/metabolismo , Cerebelo/metabolismo , Hipocampo/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neurônios/metabolismo , Potenciais de Ação , Motivos de Aminoácidos , Animais , Western Blotting , Canais de Cálcio Tipo T/genética , Linhagem Celular , Cerebelo/citologia , Imunofluorescência , Hipocampo/citologia , Humanos , Imunoprecipitação , Ativação do Canal Iônico , Cinética , Camundongos , Proteínas dos Microfilamentos/genética , Mutação , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Regulação para CimaAssuntos
Doenças Fetais/diagnóstico , Tumor Neuroectodérmico Melanótico/diagnóstico , Neoplasias Orbitárias/diagnóstico , Adulto , Cesárea , Diagnóstico Diferencial , Feminino , Doenças Fetais/diagnóstico por imagem , Doenças Fetais/patologia , Humanos , Lactente , Imageamento por Ressonância Magnética/métodos , Masculino , Tumor Neuroectodérmico Melanótico/diagnóstico por imagem , Tumor Neuroectodérmico Melanótico/patologia , Neoplasias Orbitárias/diagnóstico por imagem , Neoplasias Orbitárias/patologia , Gravidez , Doenças Raras , Tomografia Computadorizada por Raios X , Ultrassonografia Pré-Natal/métodosRESUMO
The 15-megabase pair Saccharomyces cerevisiae and the 4.7-megabase pair Escherichia coli genomes were completely cleaved at a single predetermined site by means of the Achilles' heel cleavage (AC) procedure. The symmetric lac operator (lacOs) was introduced into the circular Escherichia coli genome and into one of the 16 yeast chromosomes. Intact chromosomes from the resulting strains were prepared in agarose microbeads and methylated with Hha I (5'-GCGC) methyltransferase (M.Hha I) in the presence of lac repressor (LacI). All Hae II sites (5'-[sequence: see text]) with the exception of the one in lacOs, which was protected by LacI, were modified and thus no longer recognized by Hae II. After inactivation of M.Hha I and LacI, Hae II was used to completely cleave the chromosomes specifically at the inserted lacOs. These experiments demonstrate the feasibility of using the AC approach to efficiently extend the specificity of naturally occurring restriction enzymes and create new tools for the mapping and precise molecular dissection of multimegabase genomes.
Assuntos
DNA Bacteriano/metabolismo , DNA Fúngico/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/genética , Genes Bacterianos , Genes Fúngicos , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Bases , DNA Bacteriano/genética , DNA Fúngico/genética , Hidrólise , Dados de Sequência Molecular , Óperon , Especificidade por SubstratoRESUMO
Mapping and manipulation of very large genomes, including the human genome, would be facilitated by the availability of a DNA cleavage method with very high site specificity. Therefore, a general method was devised that extends the effective recognition sequences well beyond the present 8-base pair limit by combining the specificity of the restriction endonuclease with that of another sequence-specific protein that binds tightly to DNA. It was shown that the tightly binding lac or lambda repressor protects a restriction site within the operator from specific modification methylases, M.Hha I or M.Hph I, while all other similar sites are methylated and thus rendered uncleavable. A plasmid containing a symmetric lac operator was specifically cleaved by Hha I, only at the site within the operator, after M.Hha I methylation in the presence of the lac repressor, whereas the remaining 31 Hha I sites on this plasmid were methylated and thus not cleaved. Analogous results were obtained with the Hae II site within the lac operator, which was similarly protected by the lac repressor, and with the Hph I site within the phage lambda oL operator, which was protected by lambda repressor from M.Hph I methylation.
Assuntos
Enzimas de Restrição do DNA/metabolismo , DNA/metabolismo , Regiões Operadoras Genéticas , Bacteriófago lambda/análise , Bacteriófago lambda/genética , Temperatura Alta , Óperon Lac , Metilação , Plasmídeos , Proteínas Repressoras/metabolismo , Especificidade por SubstratoRESUMO
The actin-binding protein Kelch-like 1 (KLHL1) is a neuronal protein that belongs to the evolutionarily-conserved Kelch protein super-family. The mammalian KLHL1 is brain-specific, cytosolic and can form multimers and bind actin filaments. KLHL1's function is likely that of an actin-organizing protein, possibly modulating neurite outgrowth, the dynamic morphology of dendritic spine heads; or anchoring proteins essential for post-synaptic function, like ion channels. Targeted deletion of the KLHL1 gene in Purkinje neurons results in dendritic deficits in these neurons, abnormal gait, and progressive loss of motor coordination in mice [He Y, Zu T, Benzow KA, Orr HT, Clark HB, Koob MD (2006) Targeted deletion of a single SCA8 ataxia locus allele in mice causes abnormal gait, progressive loss of motor coordination, and Purkinje cell dendritic deficits. J Neurosci 26:9975-9982]. Here we tested the hypothesis that KLHL1 may interact and modulate voltage-gated calcium channels by assessing the interaction of the principal subunit of P/Q-type channels, alpha(1A), with KLHL1. Experiments in human embryonic kidney line HEK 293 (HEK) cells and cerebellar primary cultures revealed co-incidence of alpha(1A) and KLHL1 immunoreactivity when testing both the endogenous or epitope-tagged versions of the proteins. Similarly, co-immunoprecipitation experiments in HEK cells and brain tissue exposed the presence of KLHL1 in protein samples immunoprecipitated with FLAG-tagged or alpha(1A) antibodies. Functional studies of KLHL1 on P/Q-type current properties probed with whole-cell patch clamp revealed a significant increase in mean current density in the presence of KLHL1 (80% increase; from -13.2+/-2.0 pA/pF to -23.7+/-4.2 pA/pF, P<0.02), as well as a shift in steady state activation V(50) of -5.5 mV (from 12.8+/-1.8 mV to 7.3+/-1.0 mV, P<0.02). Our data are consistent with a modulatory effect of KLHL1 on the P/Q-type calcium channel function and suggest a possible novel role for KLHL1 in cellular excitability.
Assuntos
Canais de Cálcio Tipo N/fisiologia , Cerebelo/fisiologia , Proteínas dos Microfilamentos/fisiologia , Neurônios/fisiologia , Potenciais de Ação/fisiologia , Cálcio , Linhagem Celular , Células Cultivadas , DNA Complementar/genética , Dendritos/fisiologia , Dendritos/ultraestrutura , Humanos , Rim , Cinética , Proteínas dos Microfilamentos/genética , Neuritos/fisiologia , Ataxias Espinocerebelares , Sinapses/fisiologia , TransfecçãoRESUMO
Intrarachidian cystic lesions are frequent, with highly varied causes. They can be classified according to their location into intramedullary cystic lesions and extramedullary cystic lesions. In these two categories, they can then be regrouped according to the tissue from which they develop. MRI is the first-choice examination for the study of the intracanal contents and the differential diagnosis between the various lesions.
Assuntos
Cistos do Sistema Nervoso Central/diagnóstico , Cistos/diagnóstico , Imageamento por Ressonância Magnética , Doenças da Medula Espinal/diagnóstico , Neoplasias da Medula Espinal/diagnóstico , Adulto , HumanosRESUMO
The development of post-processing reconstruction techniques has opened new possibilities for the study of in-utero fetal brain MRI data. Recent cortical surface analysis have led to the computation of quantitative maps characterizing brain folding of the developing brain. In this paper, we describe a novel feature selection-based approach that is used to extract the most discriminative and sparse set of features of a given dataset. The proposed method is used to sparsely characterize cortical folding patterns of an in-utero fetal MR dataset, labeled with heterogeneous gestational age ranging from 26 weeks to 34 weeks. The proposed algorithm is validated on a synthetic dataset with both linear and non-linear dynamics, supporting its ability to capture deformation patterns across the dataset within only a few features. Results on the fetal brain dataset show that the temporal process of cortical folding related to brain maturation can be characterized by a very small set of points, located in anatomical regions changing across time. Quantitative measurements of growth against time are extracted from the set selected features to compare multiple brain regions (e.g. lobes and hemispheres) during the considered period of gestation.
Assuntos
Algoritmos , Encéfalo/diagnóstico por imagem , Encéfalo/embriologia , Imageamento por Ressonância Magnética/métodos , Encéfalo/anatomia & histologia , HumanosRESUMO
The management of sporadic late-onset cerebellar ataxias represents a very heterogeneous group of patients and remains a challenge for neurologist in clinical practice. We aimed at describing the different causes of sporadic late-onset cerebellar ataxias that were diagnosed following standardized, exhaustive investigations and the population characteristics according to the aetiologies as well as at evaluating the relevance of these investigations. All patients consecutively referred to our centre due to sporadic, progressive cerebellar ataxia occurring after 40 years of age were included in the prospective, observational study. 80 patients were included over a 2 year period. A diagnosis was established for 52 patients (65%) corresponding to 18 distinct causes, the most frequent being cerebellar variant of multiple system atrophy (n = 29). The second most frequent cause was inherited diseases (including spinocerebellar ataxias, late-onset Friedreich's disease, SLC20A2 mutations, FXTAS, MELAS, and other mitochondrial diseases) (n = 9), followed by immune-mediated or other acquired causes. The group of patient without diagnosis showed a slower worsening of ataxia (p < 0.05) than patients with multiple system atrophy. Patients with later age at onset experienced faster progression of ataxia (p = 0.001) and more frequently parkinsonism (p < 0.05) than patients with earlier onset. Brain MRI, DaT scan, genetic analysis and to some extent muscle biopsy, thoracic-abdominal-pelvic tomodensitometry, and cerebrospinal fluid analysis were the most relevant investigations to explore sporadic late-onset cerebellar ataxia. Sporadic late-onset cerebellar ataxias should be exhaustively investigated to identify the underlying causes that are numerous, including inherited causes, but dominated by multiple system atrophy.
Assuntos
Ataxia Cerebelar/diagnóstico , Ataxia Cerebelar/etiologia , Atrofia de Múltiplos Sistemas/complicações , Adulto , Idade de Início , Idoso , Encéfalo/diagnóstico por imagem , Canais de Cálcio/genética , Ataxia Cerebelar/genética , Ataxia Cerebelar/patologia , Eletromiografia , Feminino , Ataxia de Friedreich/complicações , Humanos , Estudos Longitudinais , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Atrofia de Múltiplos Sistemas/diagnóstico por imagem , Mutação/genética , Condução Nervosa/fisiologia , Exame Neurológico , Proteínas Proto-Oncogênicas c-sis/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptores Acoplados a Proteínas G/genética , Receptores Virais/genética , Estudos Retrospectivos , Índice de Gravidade de Doença , Ataxias Espinocerebelares/complicações , Estatísticas não Paramétricas , Receptor do Retrovírus Politrópico e XenotrópicoRESUMO
AIMS: To compare unenhanced, gadolinium enhanced, delayed gadolinium enhanced FLAIR images, gadolinium enhanced and delayed gadolinium enhanced T1 images in different types of leptomeningeal diseases, and to determine the most accurate MRI sequence for the diagnosis of leptomeningeal disease. MATERIAL: and methods: Ten patients (6 men, 4 women, age: 52,7+/-16,4) clinically suspected of cerebral leptomeningeal infectious or tumoral disease underwent brain MR examination: Axial FLAIR and T1 SE images were acquired before, immediately after administration of gadobenate dimeglumine (0.1 mmol per kilogram of body weight) (early enhancement), and 20 minutes after injection of contrast media (delayed enhancement). Images were analysed to determine the more appropriate technique for the diagnosis of leptomeningeal disease. RESULTS: Early enhanced FLAIR and delayed enhanced T1 were always more or equally accurate for the diagnosis of leptomeningeal diasease, as compared to, respectively, unenhanced FLAIR and early enhanced T1 images Delayed enhanced FLAIR was always more accurate for the diagnosis of leptomeningeal disease as compared to early enhanced FLAIR images. Delayed enhanced FLAIR was in most of the cases more accurate for the diagnosis of leptomeningeal disease as compared to delayed enhanced T1 images. CONCLUSION: Delayed enhanced FLAIR MR sequence seems to improve the diagnosis of leptomeningeal infectious or tumoral diseases as compared to other MR sequences.
Assuntos
Imageamento por Ressonância Magnética/métodos , Neoplasias Meníngeas/diagnóstico , Meningites Bacterianas/diagnóstico , Meningite Viral/diagnóstico , Adolescente , Idoso , Meios de Contraste , Infecções por Vírus Epstein-Barr/diagnóstico , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Infecções Estreptocócicas/diagnóstico , Streptococcus agalactiaeRESUMO
The authors describe normal imaging of the meninges and meningeal spaces and MR (magnetic resonance) imaging findings in tumoral and nontumoral diseases. Dural or/and pial enhancement may be related to tumoral, infectious or granulomatous diseases.
Assuntos
Doenças do Sistema Nervoso Central/diagnóstico , Meios de Contraste , Imageamento por Ressonância Magnética/métodos , Neoplasias Meníngeas/diagnóstico , Meninges/anatomia & histologia , Infecções do Sistema Nervoso Central/diagnóstico , Dura-Máter/patologia , Humanos , Pia-Máter/patologiaRESUMO
A novel technique for the creation of rare restriction sites was described by Koob et al. [Science 241 (1988) 1084-1086]. This technique, Achilles' heel cleavage (AC), relies on the use of a bound repressor molecule to protect only one of many identical restriction sites from a modification methyltransferase that inactivates all other restriction sites. The technique was applied to a small plasmid and shown to work efficiently with two repressor/operator systems: lac repressor/lacO operator and lambda repressor/lambda oL1 operator. Here, we have extended these results to a lac operator carried by a much larger vector, namely a 44-kb phage lambda construct. In addition, we have evaluated the effect of altering the stability of the lac repressor/lac operator complex by varying both the operator and the repressor. We have also evaluated several more restriction/modification systems (MboI, Dam, MspI and AluI) in addition to HhaI and HaeII used earlier. Finally, we extended the AC technique to a third system, that of the phage 434 repressor and a synthetic 434 operator. From our results we conclude that the AC method should be applicable to the mapping of large genomes and to measuring the strength of operator-repressor interactions. AC could also be applied to identifying and evaluating many different DNA-binding proteins and their sites of action.
Assuntos
Bacteriófago lambda/genética , Enzimas de Restrição do DNA/metabolismo , Proteínas Repressoras/metabolismo , Mapeamento por Restrição , Fatores de Transcrição/metabolismo , Sequência de Bases , Óperon Lac , Dados de Sequência Molecular , Regiões Operadoras GenéticasRESUMO
We have constructed a plasmid system designed for the insertion of cloned DNA (e.g., genes, gene fusions, regulatory elements, etc.) into the Escherichia coli genome. Its principal feature is the presence of two tandem lox sites on the plasmids, which upon Cre-mediated in vitro recombination resolve the plasmids into ori- and ori+ DNA circles. The non-replicating ori- circles contain the lambda attP site, several unique restriction sites for cloning, a NotI site and KmR, a kanamycin-resistance-encoding gene. The ori+ circles carry the origin of DNA replication (ori) together with several cleavage sites not present in the ori- circles, including the rare site for the very efficient I-SceI enzyme, that are used to inactivate the ori+ circles and any unresolved plasmid DNA. We have used this system to insert cloned DNA into the host genome at (i) the attB site, by Int-mediated integration and (ii) at any predetermined sequence, as mediated by the Rec system(s) of the host. The genomes of the resulting transformants were analyzed by NotI digestion of the chromosomal DNA, embedded in agarose microbeads, followed by pulsed-field gel electrophoresis. A system for the retrieval of DNA fragments inserted at the attB site was also developed.
Assuntos
DNA Nucleotidiltransferases/metabolismo , Escherichia coli/genética , Marcação de Genes , Integrases , Plasmídeos , Origem de Replicação , Proteínas Virais , Sequência de Bases , DNA Recombinante , Galactoquinase/genética , Genes Bacterianos , Dados de Sequência Molecular , Recombinação GenéticaRESUMO
LacI-mediated Achilles' cleavage (AC) is a method for selective fragmentation of chromosomes at special lac operator sites introduced by gene targeting methods [Koob and Szybalski, Science 250 (1990) 271-273]. The Saccharomyces cerevisiae APA1 gene, coding for diadenosine 5', 5"'-P1, P4-tetraphosphate phosphorylase I, has previously been shown to be located on chromosome III [Kaushal et al., Gene 95 (1990) 79-84]. We have now used the AC method to map APA1 gene to a site 44 kb from the left terminus of the chromosome, between the HIS4 and HML genes. This location was confirmed by the comparison of restriction maps of the APA1 gene region to published restriction maps of chromosome III.
Assuntos
Nucleotidiltransferases/genética , Saccharomyces cerevisiae/genética , Eletroforese em Gel de Campo Pulsado , Genes Fúngicos , Técnicas Genéticas , Hibridização de Ácido Nucleico , Nucleotidiltransferases/metabolismo , Plasmídeos , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologiaRESUMO
Integration host factor (IHF)-mediated protection against enzymatic methylation at ihf-overlapping sites provides the basis for this novel application of the Achilles' cleavage (AC) technique [Koob et al., Science 241 (1988) 1084-1086] for generating rare natural cleavage sites. When applying IHF-AC to plasmid, phage lambda, Escherichia coli and yeast genomes, only a few of the EcoRI, HinfI, and MboI sites (which overlapped the ihf sites) remained cleavable after prior methylation with the cognate M.EcoRI, M.HinfI, or Dam methyltransferases in the presence of IHF. Thus, IHF-AC essentially converted these enzymes into very rare cutters. The extent of cleavage could be controlled by varying the IHF:DNA ratio and temperature. Moreover, the method permits the genomic location and strength of the ihf sites to be determined.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Mapeamento por Restrição , Proteínas de Bactérias/genética , Bacteriófago lambda/genética , Sequência de Bases , Proteínas de Ligação a DNA/genética , Desoxirribonuclease EcoRI , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/genética , Genoma Bacteriano , Genoma Fúngico , Fatores Hospedeiros de Integração , Metilação , Dados de Sequência Molecular , Saccharomyces cerevisiae/genéticaRESUMO
OBJECTIVE: To determine the incidence of spinocerebellar ataxia (SCA) types 1, 2, 3, 6, and 7 and Friedreich's ataxia (FA) among a large panel of ataxia families. BACKGROUND: The ataxias are a clinically and genetically heterogeneous group of neurodegenerative diseases that variably affect the cerebellum, brainstem, and spinocerebellar tracts. Trinucleotide repeat expansions have been shown to be the mutational mechanism for five dominantly inherited SCAs as well as FA. METHODS: We collected DNA samples and clinical data from patients representing 361 families with adult-onset ataxia of unknown etiology. Patients with a clinical diagnosis of FA were specifically excluded from our collection. RESULTS: Among the 178 dominant kindreds, we found SCA1 expansion at a frequency of 5.6%, SCA2 expansion at a frequency of 15.2%, SCA3 expansion at a frequency of 20.8%, SCA6 expansion at a frequency of 15.2%, and SCA7 expansion at a frequency of 4.5%. FA alleles were found in 11.4% of apparently recessive and 5.2% of apparently sporadic patients. Among these patients the repeat sizes for one or both FA alleles were relatively small, with sizes for the smaller allele ranging from 90 to 600 GAA repeats. The clinical presentation for these patients is atypical for FA, with one or more of the following characteristics: adult onset of disease, retained tendon reflexes, normal plantar response, and intact or partially intact sensory perceptions. CONCLUSIONS: Pathogenic trinucleotide repeat expansions were found among 61% of the dominant kindreds. Among patients with apparently recessive or negative family histories of ataxia, 6.8% and 4.4% tested positive for a CAG expansion at one of the dominant loci, and 11.4 and 5.2% of patients with apparently recessive or sporadic forms of ataxia had FA expansions. Because of the significant implications that a dominant versus recessive inheritance pattern has for future generations, it is important to screen patients who do not have a clearly dominant inheritance pattern for expansions at both the FA and the dominant ataxia loci.
Assuntos
Saúde da Família , Ataxia de Friedreich/epidemiologia , Ataxia de Friedreich/genética , Repetições de Trinucleotídeos , Adulto , DNA/análise , Genes Dominantes , Genes Recessivos , Homozigoto , Humanos , Incidência , Pessoa de Meia-IdadeRESUMO
Several halogenated alkenes are nephrotoxic; some others induce renal tubular adenocarcinomas in rodents after lifelong administration. A bioactivation mechanism accounting for the organ-selective tumor induction has been elucidated: conjugation of the parent compounds with glutathione (GSH), catalyzed by hepatic GSH S-transferases, results in the formation of haloalkyl and halovinyl glutathione S-conjugates. Formation of S-conjugates (identified by NMR and mass spectrometry) could be demonstrated with trichloroethene, tetrachloroethene, hexachlorobutadiene, perfluoropropene, trichlorotrifluoropropene, and dichloroacetylene in incubations with rat liver microsomes and in the isolated perfused rat liver. The GSH conjugates formed are eliminated from the rat liver with the bile and may be translocated to the kidney, intact or after metabolism to the corresponding cysteine S-conjugates that are metabolized in the kidney by renal tubular cysteine conjugate beta-lyase (beta-lyase) to reactive intermediates, most likely thioacylchlorides and thioketenes. Interaction of these potent electrophiles with DNA [demonstrated for intermediates formed from S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine] causes mutagenicity in bacteria, genotoxicity in cultured renal cells, and cytotoxicity in kidney cells. As an alternative to beta-lyase-catalyzed cleavage, the cysteine S-conjugates may be acetylated to the corresponding mercapturic acids, which have been identified in urine. The ability of the kidney to concentrate GSH and cysteine S-conjugates and the intensive metabolism of GSH S-conjugates to cysteine S-conjugates in this organ are evidently responsible for the organotropic carcinogenicity.