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OBJECTIVES: Our objective was to reinforce clinical knowledge of hearing impairment in KBG syndrome. KBG syndrome is a rare genetic disorder due to monoallelic pathogenic variations of ANKRD11.The typical phenotype includes facial dysmorphism, costal and spinal malformation and developmental delay. Hearing loss in KBG patients has been reported for many years, but no study has evaluated audiological phenotyping from a clinical and an anatomical point of view. METHODS: This French multicenter study included 32 KBG patients with retrospective collection of data on audiological features, ear imaging and genetic investigations. RESULTS: We identified a typical audiological profil in KBG syndrome: conductive (71%), bilateral (81%), mild to moderate (84%) and stable (69%) hearing loss, with some audiological heterogeneity. Among patients with an abnormality on CT imaging (55%), ossicular chain impairment (67%), fixation of the stapes footplate (33%) and inner-ear malformations (33%) were the most common abnormalities. CONCLUSION: We recommend a complete audiological and radiological evaluation and an ENT-follow up in all patients presenting with KBG Syndrome. Imaging evaluation is necessary to determine the nature of lesions in the middle and inner ear.
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Anormalidades Múltiplas , Doenças do Desenvolvimento Ósseo , Surdez , Deficiência Intelectual , Anormalidades Dentárias , Humanos , Anormalidades Múltiplas/genética , Deficiência Intelectual/genética , Doenças do Desenvolvimento Ósseo/genética , Anormalidades Dentárias/genética , Fácies , Estudos Retrospectivos , Proteínas Repressoras/genética , FenótipoRESUMO
INTRODUCTION: Inflammatory Breast Cancer (IBC) is the most aggressive form of breast carcinoma characterized by rapid onset of inflammatory signs and its molecular fingerprint has not yet been elucidated. OBJECTIVES: The objective of this study was to detect both gene expression levels and alternate RNA splice variants specific for IBC. METHODS: W e performed splice-sensitive array profiling using Affymetrix Exon Array and quantitative RT-PCR analyses in 177 IBC compared to 183 non-IBC. We also assessed the prognostic value of the identified candidate genes and splice variants. RESULTS: A 5-splice signature (HSPA8, RPL10, RPL4, DIDO1 and EVL) was able to distinguish IBC from non-IBC tumors (p<10-7). This splice signature was associated with poor metastasis-free survival in hormone receptor-negative non-IBC (p=0.02), but had no prognostic value in IBC. PAM analysis of dysregulated genes in IBC compared to non-IBC identified a 10-gene signature highly predictive of IBC phenotype and conferring a poor prognosis in non-IBC. The genes most commonly upregulated in IBC were 3 hemoglobin genes able to reliably discriminate IBC from non-IBC (p<10-4). Hb protein expression in epithelial breast tumor cells was confirmed by immunohistochemistry. CONCLUSION: IBC has a specific spliced transcript profile and is characterized by hemoglobin gene overexpression that should be investigated in further functional studies.
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The antiretroviral agent nelfinavir has antimyeloma activity and can overcome resistance to bortezomib. Our phase I/II trial investigated whether adding nelfinavir to lenalidomide-dexamethasone can overcome lenalidomide resistance in lenalidomide-refractory multiple myeloma (MM). Twenty-nine patients were included (high-risk cytogenetic aberrations 31%; ≥2 prior therapy lines 93%; lenalidomide-bortezomib double-refractory 34%). Twenty-four patients (83%) had prior bortezomib and 10 (34%) were lenalidomide-bortezomib double-refractory. They received four cycles of nelfinavir 2500 mg/day with standard-dose lenalidomide (25 mg days 1-21) and dexamethasone (40/20 mg days 1, 8, 15, 22). Minor response or better was achieved in 16 patients (55%; 95% CI 36-74%), including 40% of those who were lenalidomide-bortezomib double-refractory, and partial response or better in nine patients (31%; 95% CI 15-51%). Median progression-free survival was 3.4 (95% CI 2.0-4.9) months and median overall survival 21.6 (13.0-50.1) months. Lenalidomide-related pneumonitis, pneumonia, and neutropenic fever occurred, but there were no unexpected adverse events. Peripheral blood mononuclear cells showed a 45% (95% CI 40-51%) reduction in total proteasome activity from baseline and significant induction of unfolded protein response and autophagy. Thus, nelfinavir-lenalidomide-dexamethasone is an active oral combination in lenalidomide-refractory MM.
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Anti-Inflamatórios/uso terapêutico , Dexametasona/uso terapêutico , Inibidores da Protease de HIV/uso terapêutico , Lenalidomida/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Nelfinavir/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anti-Inflamatórios/farmacologia , Dexametasona/farmacologia , Feminino , Inibidores da Protease de HIV/farmacologia , Humanos , Lenalidomida/farmacologia , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Nelfinavir/farmacologiaRESUMO
OBJECTIVE: To investigate the obstetric outcome of women carriers of the oxidative phosphorylation (OXPHOS) disorder mutation. DESIGN: A retrospective cohort study in a single tertiary centre. SETTING: A review of the obstetric history of women referred for prenatal screening of a mitochondrial disorder was performed. POPULATION: Women were divided into three groups: (1) women carrying mitochondrial DNA (mtDNA) mutations; (2) healthy women with a family history of mtDNA-related OXPHOS disorder; and (3) healthy women carrying heterozygote nuclear DNA mutations. METHODS: Obstetric history and pregnancy complications were evaluated separately in the three groups and compared with the control group. MAIN OUTCOME MEASURES PREGNANCY COMPLICATIONS. RESULTS: Seventy-five women were included with 287 cumulative pregnancies. Groups 1 and 3 had a significantly greater proportion of terminations of pregnancy (20 and 13% versus 0.8%, P < 0.001), and a lower percentage of live births (52 and 72% versus 87%, P = 0.001), compared with controls. Apart from this, the rate of obstetric complications in group 3 did not differ from the controls. The obstetric history of women in group 1 was marked by higher rates of early miscarriages (26 versus 11%, P = 0.004), gestational diabetes (14 versus 3%, P = 0.02), intrauterine growth restriction (IUGR, 10 versus 1%, P = 0.008), and postpartum haemorrhage than were reported for controls (12 versus 2%, P = 0.01). CONCLUSION: Women who are heteroplasmic for OXPHOS mutations have a higher incidence of pregnancy losses, gestational diabetes, IUGR, and post postpartum haemorrhage. TWEETABLE ABSTRACT: Women heteroplasmic for mitochondrial DNA mutations have a higher incidence of obstetric complications, compared with the control group.
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Aborto Induzido/estatística & dados numéricos , Doenças Mitocondriais/genética , Complicações na Gravidez/genética , Diagnóstico Pré-Natal , Adulto , Feminino , França/epidemiologia , Aconselhamento Genético , Humanos , Incidência , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/epidemiologia , Gravidez , Complicações na Gravidez/diagnóstico , Complicações na Gravidez/epidemiologia , Diagnóstico Pré-Natal/estatística & dados numéricos , Estudos Retrospectivos , Atenção Terciária à SaúdeRESUMO
Wiedemann-Steiner syndrome (WSS) is a rare syndromic condition in which intellectual disability (ID) is associated with hypertrichosis cubiti, short stature, and characteristic facies. Following the identification of the causative gene (KMT2A) in 2012, only 31 cases of WSS have been described precisely in the literature. We report on 33 French individuals with a KMT2A mutation confirmed by targeted gene sequencing, high-throughput sequencing or exome sequencing. Patients' molecular and clinical features were recorded and compared with the literature data. On the molecular level, we found 29 novel mutations. We observed autosomal dominant transmission of WSS in 3 families and mosaicism in one family. Clinically, we observed a broad phenotypic spectrum with regard to ID (mild to severe), the facies (typical or not of WSS) and associated malformations (bone, cerebral, renal, cardiac and ophthalmological anomalies). Hypertrichosis cubiti that was supposed to be pathognomonic in the literature was found only in 61% of our cases. This is the largest series of WSS cases yet described to date. A majority of patients exhibited suggestive features, but others were less characteristic, only identified by molecular diagnosis. The prevalence of WSS was higher than expected in patients with ID, suggesting than KMT2A is a major gene in ID.
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Deficiência Intelectual/diagnóstico , Deficiência Intelectual/etiologia , Adolescente , Substituição de Aminoácidos , Criança , Pré-Escolar , Suscetibilidade a Doenças , Feminino , França , Sequenciamento de Nucleotídeos em Larga Escala , Histona-Lisina N-Metiltransferase/genética , Humanos , Imageamento por Ressonância Magnética , Masculino , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Fenótipo , Síndrome , Tomografia Computadorizada por Raios XRESUMO
In animal models of autism spectrum disorder (ASD), the NKCC1 chloride-importer inhibitor bumetanide restores physiological (Cl-)i levels, enhances GABAergic inhibition and attenuates electrical and behavioral symptoms of ASD. In an earlier phase 2 trial; bumetanide reduced the severity of ASD in children and adolescents (3-11 years old). Here we report the results of a multicenter phase 2B study primarily to assess dose/response and safety effects of bumetanide. Efficacy outcome measures included the Childhood Autism Rating Scale (CARS), the Social Responsive Scale (SRS) and the Clinical Global Impressions (CGI) Improvement scale (CGI-I). Eighty-eight patients with ASD spanning across the entire pediatric population (2-18 years old) were subdivided in four age groups and randomized to receive bumetanide (0.5, 1.0 or 2.0 mg twice daily) or placebo for 3 months. The mean CARS value was significantly improved in the completers group (P: 0.015). Also, 23 treated children had more than a six-point improvement in the CARS compared with only one placebo-treated individual. Bumetanide significantly improved CGI (P: 0.0043) and the SRS score by more than 10 points (P: 0.02). The most frequent adverse events were hypokalemia, increased urine elimination, loss of appetite, dehydration and asthenia. Hypokalemia occurred mainly at the beginning of the treatment at 1.0 and 2.0 mg twice-daily doses and improved gradually with oral potassium supplements. The frequency and incidence of adverse event were directly correlated with the dose of bumetanide. Therefore, bumetanide improves the core symptoms of ASD and presents a favorable benefit/risk ratio particularly at 1.0 mg twice daily.
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Transtorno do Espectro Autista/tratamento farmacológico , Bumetanida/administração & dosagem , Inibidores de Simportadores de Cloreto de Sódio e Potássio/administração & dosagem , Adolescente , Anorexia/induzido quimicamente , Astenia/induzido quimicamente , Transtorno do Espectro Autista/fisiopatologia , Transtorno do Espectro Autista/psicologia , Bumetanida/uso terapêutico , Criança , Pré-Escolar , Desidratação/induzido quimicamente , Relação Dose-Resposta a Droga , Feminino , Humanos , Hipopotassemia/induzido quimicamente , Masculino , Inibidores de Simportadores de Cloreto de Sódio e Potássio/uso terapêutico , Resultado do TratamentoRESUMO
Duplication of the Xq28 region, involving MECP2 (dupMECP2), has been primarily described in males with severe developmental delay, spasticity, epilepsy, stereotyped movements and recurrent infections. Carrier mothers are usually asymptomatic with an extremely skewed X chromosome inactivation (XCI) pattern. We report a series of six novel symptomatic females carrying a de novo interstitial dupMECP2, and review the 14 symptomatic females reported to date, with the aim to further delineate their phenotype and give clues for genetic counselling. One patient was adopted and among the other 19 patients, seven (37%) had inherited their duplication from their mother, including three mildly (XCI: 70/30, 63/37, 100/0 in blood and random in saliva), one moderately (XCI: random) and three severely (XCI: uninformative and 88/12) affected patients. After combining our data with data from the literature, we could not show a correlation between XCI in the blood or duplication size and the severity of the phenotype, or explain the presence of a phenotype in these females. These findings confirm that an abnormal phenotype, even severe, can be a rare event in females born to asymptomatic carrier mothers, making genetic counselling difficult in couples at risk in terms of prognosis, in particular in prenatal cases.
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Duplicação Gênica , Deficiência Intelectual/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Proteína 2 de Ligação a Metil-CpG/genética , Adolescente , Adulto , Criança , Cromossomos Humanos X/genética , Feminino , Aconselhamento Genético , Humanos , Deficiência Intelectual/fisiopatologia , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/fisiopatologia , Linhagem , FenótipoRESUMO
PURPOSE: To assess long-term neurodevelopmental outcome in children with hydrocephalus requiring neurosurgical treatment during the neonatal period. METHODS: This prospective longitudinal population-based study included 43 children with neonatal shunted hydrocephalus. The 43 children were prospectively reviewed in the presence of their parents at the outpatient clinic. Cognitive and motor outcomes were assessed respectively using different Wechsler scales according to age and Gross Motor Function Classification System (GMFCS). Postoperative MRI was routinely performed. RESULTS: The mean gestational age at birth of the 43 consecutive children with neonatal hydrocephalus (sex ratio M/F: 1.39) was 34.5±5.4 weeks of gestation. At mean follow-up of 10.4±4 years, mean total IQ was 73±27.7, with equivalent results in mean verbal and mean performance IQ. Of the 33 children with IQ evaluation, 18 presented an IQ≥85 (41.9%). Efficiency in walking without a mobility device (GMFCS≤2) was obtained in 37 children (86%). Only severity of postoperative ventricular dilation was significantly associated with unfavorable outcome (Evans index>0.37; odds ratio: 0.16, P=0.03). CONCLUSION: This information could be provided to those families concerned who often experience anxiety when multi-disciplinary management of neonatal hydrocephalus is required.
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Hidrocefalia/cirurgia , Doenças do Prematuro/cirurgia , Transtornos do Neurodesenvolvimento/etiologia , Complicações Pós-Operatórias/prevenção & controle , Derivação Ventriculoperitoneal , Ventriculostomia , Anormalidades Múltiplas , Neoplasias do Ventrículo Cerebral/complicações , Neoplasias do Ventrículo Cerebral/cirurgia , Feminino , Seguimentos , Idade Gestacional , Humanos , Hidrocefalia/complicações , Hidrocefalia/diagnóstico por imagem , Recém-Nascido , Recém-Nascido Prematuro , Deficiência Intelectual/epidemiologia , Deficiência Intelectual/etiologia , Deficiência Intelectual/prevenção & controle , Imageamento por Ressonância Magnética , Masculino , Transtornos dos Movimentos/epidemiologia , Transtornos dos Movimentos/etiologia , Transtornos dos Movimentos/prevenção & controle , Transtornos do Neurodesenvolvimento/epidemiologia , Transtornos do Neurodesenvolvimento/prevenção & controle , Complicações Pós-Operatórias/diagnóstico por imagem , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Estudos Prospectivos , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
BACKGROUND: Ataxia telangiectasia mutated (ATM) is a kinase that has a central role in the maintenance of genomic integrity by activating cell cycle checkpoints and promoting repair of DNA double-strand breaks (DSB). In breast cancer, a low level of ATM was correlated with poor outcome; however, the molecular mechanism of this downregulation is still unclear. METHODS: We used qRT-PCR assay to quantify mRNA levels of ATM gene in 454 breast tumours from patients with known clinical/pathological status and outcome; reverse phase protein arrays (RPPA) were used to assess the levels of ATM and 14 proteins in 233 breast tumours. RESULTS: ATM mRNA was associated with poor metastasis-free survival (MFS) (P=0.00012) on univariate analysis. ATM mRNA and protein levels were positively correlated (P=0.00040). A low level of ATM protein was correlated with poorer MFS (P=0.000025). ATM expression at mRNA or protein levels are independent prognostic factors on multivariate analysis (P=0.00046 and P=0.00037, respectively). The ATM protein level was positively correlated with the levels of six proteins of the DSB repair pathway: H2AX (P<0.0000001), XRCC5 (P<0.0000001), NBN (P<0.0000001), Mre11 (P=0.0000029), Rad50 (P=0.0064), and TP53BP1 (P=0.026), but not with proteins involved in other pathways that are altered in cancer. Low expression of ATM protein was significantly associated with high miR-203 expression (P=0.011). CONCLUSION: We confirmed that ATM expression is an independent prognostic marker at both RNA and protein levels. We showed that alteration of ATM is involved in dysregulation of the DSB repair pathway. Finally, miR-203 may be responsible for downregulation of ATM in breast cancers.
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Proteínas Mutadas de Ataxia Telangiectasia/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Carcinoma/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/diagnóstico , Carcinoma/diagnóstico , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Feminino , Humanos , MicroRNAs/genética , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/genética , Transdução de Sinais/genéticaRESUMO
Criteria defining the involvement of severe perinatal anoxia in neonatal encephalopathy in at-term newborns at birth are stringent and are rarely all present. The simultaneous action of pre- and intrapartum factors preceding neonatal hypoxic-ischemic encephalopathy are often observed. Cooling is recommended as there is evidence that it reduces mortality without increasing major disability in survivors. It must be conducted following strict clinical and electroencephalographic criteria. Other strategies for brain protection remain difficult to establish. Follow-up must be long enough to detect cognitive deficiencies, which are frequent, even if cerebral palsy is not observed.
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Hipotermia Induzida , Hipóxia-Isquemia Encefálica/diagnóstico , Hipóxia-Isquemia Encefálica/terapia , Deficiências do Desenvolvimento/prevenção & controle , Eletroencefalografia , Feminino , Humanos , Recém-Nascido , Gravidez , Diagnóstico Pré-Natal , Fatores de Risco , Nascimento a TermoRESUMO
OBJECTIVE: Presenting our experience concerning interstitial pregnancies (IP) surgical management and to evaluate our patients' subsequent long-term fertility. PATIENTS AND METHOD: Twenty patients underwent surgical treatment of IP in our department over 15 years. In this retrospective study, we present symptoms that led to diagnosis, treatments, fertility and obstetrical outcome. RESULTS: Mean gestational age at diagnosis was 8SA, with a median BHCG rate of 7411 IU/L, and a patient mean age of 30 years. Ninety percent of patients had at least one risk factor for ectopic pregnancy. Pain or bleeding were the most common symptoms at admission, 4 patients were admitted in an hypovolemic shock status. Location of the interstitial ectopic pregnancy was discovered during surgery in 45 % of cases. Six patients had a large hemoperitoneum bigger than 1L, 5 patients had an IP of uterine stump after salpingectomy for a previous ectopic pregnancy. The most used surgical technique was in 60 % of cases the excision by Endo GIA stapling(®) with salpingectomy. Regarding fertility, 12 patients wished pregnancy in the aftermath of the intervention, 10 had at least one pregnancy, among them there is an ectopic contralateral ampullary pregnancy, and a contralateral recurrence of interstitial pregnancy. Four patients were delivered by cesarean section and 4 patients were delivered vaginally, some several times. No uterine rupture occured. DISCUSSION AND CONCLUSION: Interstitial pregnancy is a rare ectopic pregnancy. Its diagnosis is difficult and may involve maternal life-threatening and fertility. In subsequent pregnancies, the clinician has to be careful concerning the risks of interstitial pregnancy recurrence and uterine rupture.
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Gravidez Intersticial/cirurgia , Adulto , Feminino , França , Procedimentos Cirúrgicos em Ginecologia/métodos , Humanos , Infertilidade Feminina/etiologia , Gravidez , Gravidez Intersticial/diagnóstico , Recidiva , Estudos Retrospectivos , Salpingectomia , Ruptura Uterina , Adulto JovemAssuntos
Antifúngicos/efeitos adversos , Fibrose Cística/complicações , Dermatite Fototóxica/etiologia , Pirimidinas/efeitos adversos , Triazóis/efeitos adversos , Adolescente , Adulto , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergillus fumigatus/efeitos dos fármacos , Criança , Pré-Escolar , Fibrose Cística/genética , Feminino , Humanos , Masculino , Pirimidinas/uso terapêutico , Índice de Gravidade de Doença , Triazóis/uso terapêutico , Voriconazol , Adulto JovemRESUMO
We have used site-directed mutagenesis to determine whether the structural context surrounding the AUG triplet influences its ability to be selected as an initiation codon by the eukaryotic preinitiation complex. AUG triplets were introduced in a loop and stem structure naturally occurring at the midpoint of the 129-nucleotides-long 5'-untranslated region of the porcine proopiomelanocortin mRNA; one AUG triplet was inserted in the loop while another was inserted in the stem of the hairpin structure. The proopiomelanocortin cDNA and the mutant cDNAs were inserted downstream from the early promoter of an expression vector derived from simian virus 40 (SV40) and transfected into monkey kidney COS-1 cells. Analysis of the proopiomelanocortin-related peptides present in the culture medium 72 h after transfection revealed that both mutant cDNAs direct the synthesis of more proopiomelanocortin than the non-mutant cDNA. The increased translational efficiency observed with both mutants is probably due to the decreased secondary structures of the shortened 5'-untranslated region. In addition, comparison of the two mutants indicates that the mutant mRNA with the AUG triplet inserted in the loop region of the hairpin structure directs the synthesis of approximately 75% more proopiomelanocortin than the mutant mRNA with the AUG triplet inserted in the stem region of the same hairpin structure, supporting a role for the structural context in the efficiency of translational initiation.
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Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica , Pró-Opiomelanocortina/genética , RNA Mensageiro , Ribossomos/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Códon , DNA/genética , Enzimas de Restrição do DNA , Haplorrinos , Rim , Dados de Sequência Molecular , Mutação , Pró-Opiomelanocortina/biossíntese , Regiões Promotoras Genéticas , Suínos , Transcrição Gênica , TransfecçãoRESUMO
To clone the nar operon of Escherichia coli without an effective selection procedure for the nar+ phenotype, a strategy utilizing nar::Tn5 mutants was employed. Partial segments of the nar operon containing Tn5 insertions were cloned into plasmid pBR322 by using the transposon resistance character for selection. A hybrid plasmid was constructed in vitro from two of these plasmids and isolated by a procedure that involved screening a population of transformed nar(Ts) mutant TS9A for expression of thermal stable nitrate reductase activity. A detailed restriction site map of the resulting plasmid, pSR95, corresponded closely to the composite restriction endonuclease map deduced for the nar region from maps of the cloned nar::Tn5 fragments. When transformed with pSR95, wild-type strain PK27 overproduced the alpha, beta, and gamma subunits of nitrate reductase, although nitrate reductase activity was only slightly increased. The alpha and beta subunits were overproduced about 5- to 10-fold and accumulated mostly as an inactive aggregate in the cytoplasm; the gamma subunit overproduction was detected as a threefold increase in the specific content of cytochrome b555 in the membrane fraction. Functional nitrate reductase and the cytochrome spectrum associated with functional nitrate reductase were restored in the nar::Tn5 mutant EE1 after transformation with pSR95. Although the specific activity of nitrate reductase in this case was less than that of the wild type, both the alpha and beta subunits appeared to be overproduced in an inactive form. In both strains PK27(pSR95) and EE1(pSR95), the formation of nitrate reductase activity and the accumulation of inactive subunits were repressed during aerobic growth. From these observations and the accumulation of inactive subunits were repressed during aerobic growth. From these observations and the demonstration that pSR95 contains a functional nor operon that encodes the alpha, beta, gamma subunits of nitrate reductase.
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Clonagem Molecular , Escherichia coli/genética , Óperon , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Enzimas de Restrição do DNA , Elementos de DNA Transponíveis , Mutação , Nitrato Redutases/genética , Fenótipo , PlasmídeosRESUMO
The synthesis of the alpha and beta subunits of nitrate reductase by 20 chlC::Tn5 insertion mutants of Escherichia coli was determined by immune precipitation of the subunits from fractions of cell extracts. Only two of the mutants produced either subunit in detectable amounts; these two accumulated the alpha subunit, but no beta subunit. In both cases the alpha subunit was present in the cytosolic fraction, in contrast to wild-type cells, in which both subunits are present mainly in the membrane fraction. EcoRI restriction fragments containing the Tn5 inserts from five of the mutants were cloned into pBR322. The insertions were localized on two contiguous EcoRI fragments spanning a 5.6-kilobase region that overlapped the contiguous ends of the two fragments. An insertion that permitted alpha subunit formation defined one end of the 5.6-kilobase region. The results indicated that the genes encoding the alpha and beta subunits of nitrate reductase were part of a chlC (nar) operon that is transcribed in the direction alpha leads to beta.