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1.
PLoS One ; 15(1): e0227183, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923238

RESUMO

This study was conducted to estimate the prevalence of Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) in retail chicken meat and broiler chickens from the Province of Quebec, Canada, and to characterize LA-MRSA isolates. A total of 309 chicken drumsticks and thighs were randomly selected in 2013 from 43 retail stores in the Monteregie. In addition, nasal swabs and caeca samples were collected in 2013-2014 from 200 broiler chickens of 38 different flocks. LA-MRSA was not detected in broiler chickens. Fifteen LA-MRSA isolates were recovered from four (1.3%) of the 309 chicken meat samples. Multi-Locus Sequence Typing (MLST) and SCCmec typing revealed two profiles (ST398-MRSA-V and ST8-MRSA-IVa), which were distinct using pulse-field gel electrophoresis (PFGE) and microarray (antimicrobial resistance and virulence genes) analyses. In addition to beta-lactam resistance, tetracycline and spectinomycin resistance was detected in all isolates from the 3 positive samples of the ST398 profile. Southern blot hybridization revealed that the resistance genes aad(D) and lnu(A), encoding resistances to aminoglycosides and lincosamides respectively, were located on plasmid. All isolates were able to produce biofilms, but biofilm production was not correlated with hld gene expression. Our results show the presence of two separate lineages of MRSA in retail chicken meat in Quebec, one of which is likely of human origin.


Assuntos
Antibacterianos/uso terapêutico , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Meticilina/uso terapêutico , Produtos Avícolas/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/epidemiologia , Aminoglicosídeos/efeitos adversos , Aminoglicosídeos/uso terapêutico , Animais , Antibacterianos/efeitos adversos , Técnicas de Tipagem Bacteriana , Biofilmes , Southern Blotting , Galinhas , Farmacorresistência Bacteriana Múltipla/genética , Eletroforese em Gel de Campo Pulsado , Microbiologia de Alimentos , Lincosamidas/efeitos adversos , Lincosamidas/uso terapêutico , Meticilina/efeitos adversos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Prevalência , Quebeque/epidemiologia
2.
Methods Mol Biol ; 2119: 61-72, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989514

RESUMO

Neutral-neutral 2-dimensional agarose gel electrophoresis enables the detection of replication intermediate structures in DNA. Here we describe how DNA from Escherichia coli cells can be purified to retain replication intermediates and then be separated by size and shape using two consecutive agarose gel electrophoresis protocols. The DNA structures present within a localized region can be visualized by a Southern blotting/radioactive hybridisation protocol.


Assuntos
Southern Blotting , Replicação do DNA , DNA Bacteriano , Eletroforese em Gel Bidimensional , Escherichia coli , DNA Bacteriano/análise , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Eletroforese em Gel de Ágar , Escherichia coli/genética , Escherichia coli/metabolismo
3.
Anal Bioanal Chem ; 411(28): 7451-7460, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31588523

RESUMO

Cervical cancer is the second most common cancer in the world's woman population with a high incidence in developing countries where diagnostic conditions for the cancer are poor. The main culprit causing the cancer is the human papillomavirus (HPV). HPV is divided into three major groups, i.e., high-risk (HR) group, probable high-risk (pHR) group, and low-risk (LR) group according to their potential of causing cervical cancer. Therefore, developing a sensitive, reliable, and cost-effective point-of-care diagnostic method for the virus genotypes in developing countries even worldwide is of high importance for the cancer prevention and control strategies. Here we present a combined method of isothermal recombinase polymerase amplification (RPA), lateral flow dipstick (LFD), and reverse dot blot (RDB), in quick point-of-care identification of HPV genotypes. The combined method is highly specific to HPV when the conserved L1 genes are used as targeted genes for amplification. The method can be used in identification of HPV genotypes at point-of-care within 1 h with a sensitivity of low to 100 fg of the virus genomic DNA. We have demonstrated that it is an excellent diagnostic point-of-care assay in monitoring the disease without time-consuming and expensive procedures and devices.


Assuntos
Southern Blotting/métodos , Genes Virais , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sequência de Aminoácidos , DNA Viral/análise , DNA Viral/normas , Humanos , Limite de Detecção , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Reprodutibilidade dos Testes
4.
Methods Mol Biol ; 2031: 313-322, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31473968

RESUMO

Ultraviolet rays induce interstrand and intrastrand DNA cross-links, usually thymine-thymine cyclobutane dimer (T-T) and thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct (T (6-4) T). These DNA cross-links, if left unrepaired, increase the risk of these mutation being incorporated in the genetic material (i.e., DNA). Numerous studies have reported the mutagenic potential of above mentioned DNA adducts in prokaryotes, yeast and mammalian cells. Different techniques have been developed to identify such DNA adducts such as immuno-Southern blotting. This is a routinely used quantitative method to determine especially the amount of thymine dimers formed, following irradiation. In this chapter, the detailed methodology to identify thymine dimers formation is provided, using specific antibody against these adducts.


Assuntos
Southern Blotting/métodos , Dano ao DNA/efeitos da radiação , Dímeros de Pirimidina/análise , Raios Ultravioleta/efeitos adversos , Animais , Técnicas de Cultura de Células/métodos , Humanos
5.
Microbiol Res ; 226: 55-64, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284945

RESUMO

Functional association between genomic loci and specific biological traits remains lacking in many fungi, including the African tree pathogen Ceratocystis albifundus. This is mainly because of the absence of suitable transformation systems for allowing genetic manipulation of this and other fungi. Here, we present an optimized protocol for Agrobacterium tumefaciens-mediated transformation of C. albifundus. Strain AGL-1 of A. tumefaciens and four binary T-DNA vectors (conferring hygromycin B or geneticin resistance and/or expressing the green fluorescent protein [GFP]) were used for transforming germinated conidia of three isolates of C. albifundus. Stable expression of these T-DNA-encoded traits was confirmed through sequential sub-culturing of fungal transformants on selective and non-selective media and by using PCR and sequence analysis. Single-copy integration of the respective T-DNAs into the genomes of these fungi was confirmed using Southern hybridization analysis. The range of experimental parameters determined and optimised included: (i) concentrations of hygromycin B and geneticin required for inhibiting growth of the wild type fungus and (ii) the dependence of transformation on acetosyringone for inducing the bacterium's virulence genes, as well as (iii) the duration of fungus-bacterium co-cultivation periods and (iv) the concentrations of fungal conidia and bacterial cells used for the latter. The system developed in this study is stable with a high-efficiency, yielding up to 400 transformants per 106 conidia. This is the first report of a transformation protocol for C. albifundus and its availability will be invaluable for functional studies in this important fungus.


Assuntos
Agrobacterium tumefaciens/genética , Ascomicetos/genética , Transformação Genética , Ascomicetos/citologia , Ascomicetos/efeitos dos fármacos , Ascomicetos/crescimento & desenvolvimento , Southern Blotting , Carbenicilina/farmacologia , Técnicas de Cocultura , DNA Bacteriano , Regulação Fúngica da Expressão Gênica , Gentamicinas/farmacologia , Proteínas de Fluorescência Verde/genética , Higromicina B/farmacologia , Canamicina/farmacologia , Reação em Cadeia da Polimerase , Análise de Sequência , Virulência/genética
6.
Int J Parasitol ; 49(9): 725-735, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31247198

RESUMO

The malaria parasite sporozoite sequentially invades mosquito salivary glands and mammalian hepatocytes; and is the Plasmodium lifecycle infective form mediating parasite transmission by the mosquito vector. The identification of several sporozoite-specific secretory proteins involved in invasion has revealed that sporozoite motility and specific recognition of target cells are crucial for transmission. It has also been demonstrated that some components of the invasion machinery are conserved between erythrocytic asexual and transmission stage parasites. The application of a sporozoite stage-specific gene knockdown system in the rodent malaria parasite, Plasmodium berghei, enables us to investigate the roles of such proteins previously intractable to study due to their essentiality for asexual intraerythrocytic stage development, the stage at which transgenic parasites are derived. Here, we focused on the rhoptry neck protein 11 (RON11) that contains multiple transmembrane domains and putative calcium-binding EF-hand domains. PbRON11 is localised to rhoptry organelles in both merozoites and sporozoites. To repress PbRON11 expression exclusively in sporozoites, we produced transgenic parasites using a promoter-swapping strategy. PbRON11-repressed sporozoites showed significant reduction in attachment and motility in vitro, and consequently failed to efficiently invade salivary glands. PbRON11 was also determined to be essential for sporozoite infection of the liver, the first step during transmission to the vertebrate host. RON11 is demonstrated to be crucial for sporozoite invasion of both target host cells - mosquito salivary glands and mammalian hepatocytes - via involvement in sporozoite motility.


Assuntos
Anopheles/parasitologia , Hepatócitos/parasitologia , Plasmodium berghei/fisiologia , Proteínas de Protozoários/fisiologia , Animais , Southern Blotting , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Eritrócitos/parasitologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Proteínas de Protozoários/imunologia , Coelhos , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândulas Salivares/parasitologia , Esporozoítos/fisiologia
7.
BMC Plant Biol ; 19(1): 246, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182023

RESUMO

BACKGROUND: Rapid-cycling Brassica rapa (RCBr), also known as Wisconsin Fast Plants, are small robust plants with a short lifecycle that are widely used in biology teaching. RCBr have been used for decades but there are no published reports of RCBr genetic transformation. Agrobacterium-mediated vacuum infiltration has been used to transform pakchoi (Brassica rapa ssp. chinensis) and may be suitable for RCBr transformation. The floral dip transformation method, an improved version of vacuum infiltration, could make the procedure easier. RESULTS: Based on previous findings from Arabidopsis and pakchoi, plants of three different ages were inoculated with Agrobacterium. Kanamycin selection was suboptimal with RCBr; a GFP screen was used to identify candidate transformants. RCBr floral bud dissection showed that only buds with a diameter less than 1 mm carried unsealed carpels, a key point of successful floral dip transformation. Plants across a wide range of inflorescence maturities but containing these immature buds were successfully transformed, at an overall rate of 0.1% (one per 1000 T1 seeds). Transformation was successful using either vacuum infiltration or the floral dip method, as confirmed by PCR and Southern blot. CONCLUSION: A genetic transformation system for RCBr was established in this study. This will promote development of new biology teaching tools as well as basic biology research on Brassica rapa.


Assuntos
Agrobacterium/fisiologia , Brassica rapa/genética , Brassica rapa/microbiologia , Engenharia Genética/métodos , Transformação Genética , Southern Blotting , Flores/genética , Reação em Cadeia da Polimerase
8.
Adv Clin Chem ; 90: 81-132, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31122612

RESUMO

Ever since their discovery, the telomeres and the telomerase have been topics of intensive research, first as a mechanism of cellular aging and later as an indicator of health and diseases in humans. By protecting the chromosome ends, the telomeres play a vital role in preserving the information in our genome. Telomeres shorten with age and the rate of telomere erosion provides insight into the proliferation history of cells. The pace of telomere attrition is known to increase at the onset of several pathological conditions. Telomere shortening has been emerging as a potential contributor in the pathogenesis of several neurological disorders including autism spectrum disorders (ASD), schizophrenia, Alzheimer's disease (AD), Parkinson's disease (PD) and depression. The rate of telomere attrition in the brain is slower than that of other tissues owing to the low rate of cell proliferation in brain. Telomere maintenance is crucial for the functioning of stem cells in brain. Taking together the studies on telomere attrition in various neurological disorders, an association between telomere shortening and disease status has been demonstrated in schizophrenia, AD and depression, in spite of a few negative reports. But, studies in ASD and PD have failed to produce conclusive results. The cause-effect relationship between TL and neurological disorders is yet to be elucidated. The factors responsible for telomere erosion, which have also been implicated in the pathogenesis of neurological disorders, need to be explored in detail. Telomerase activation is now being considered as a potential therapeutic strategy for neurological disorders.


Assuntos
Doenças do Sistema Nervoso/genética , Telômero/genética , Telômero/metabolismo , Animais , Southern Blotting , Humanos , Hibridização in Situ Fluorescente , Reação em Cadeia da Polimerase
9.
Plant Mol Biol ; 100(6): 621-634, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31140020

RESUMO

KEY MESSAGE: An exhaustive analysis of a group of closely related parasitic plants shows a predominantly gradual reduction in plastid genome composition and provides the most reduced plastomes in the genus Cuscuta. Parasitic plants have a diminished to completely absent reliance on photosynthesis, and are characterized by sweeping morphological, physiological, and genomic changes. The plastid genome (plastome) is highly conserved in autotrophic plants but is often reduced in parasites, and provides a useful system for documenting the genomic effects of a loss of photosynthesis. Previous studies have shown a substantial degree of heterogeneity in plastome length and composition across the species of the genus Cuscuta. Specifically, species in Cuscuta sect. Ceratophorae were suspected to exhibit even more dynamic plastome evolution than the rest of the genus. This complex of eight closely related species was exhaustively sampled here, and one accession per species was sequenced via a high-throughput approach. Complete plastid genomes were assembled and annotated for each of these species and were found to be 61-87 kbp in length, representing a 45-60% reduction relative to autotrophic Convolvulaceae. The most reduced plastomes on this spectrum have lost the bulk of their photosynthetic genes and are the first fully holoparasitic plastomes described for Cuscuta. The fine-scale nature of the system introduced here allowed us to phylogenetically triangulate the locations of gene loss and pseudogenization events precisely, and to construct a step-by-step model of plastome evolution in these plants. This model reveals an intense burst of gene loss along the branch leading to the most reduced plastomes, and a few idiosyncratic changes elsewhere, allowing us to conclude that the tempo of plastid evolution in sect. Ceratophorae is a blend of gradual and punctuated mode.


Assuntos
Cuscuta/genética , Genomas de Plastídeos , Plantas/parasitologia , Plastídeos/genética , Southern Blotting , DNA de Plantas/genética , Evolução Molecular , Genoma de Planta , Fotossíntese/genética , Filogenia , Pseudogenes , Análise de Sequência de DNA
11.
Plant Sci ; 283: 407-415, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128711

RESUMO

Starch content and composition are major determinants of yield and quality in maize. In recent years, the major genes for starch metabolism have been cloned in this species. However, the role of transcription factors in regulating the starch metabolism pathway remains unclear. The ZmbZIP22 gene encodes a bZIP transcription factor. In our study, plants overexpressing ZmbZIP22 showed reductions in the size of starch granules, the size and weight of seeds, reduced amylose content, and alterations in the chemical structure of starch granules. Also, overexpression of ZmbZIP22 resulted in increases in the contents of soluble sugars and reducing sugars in transgenic rice and maize. ZmbZIP22 promotes the transcription of starch metabolism genes by binding to their promoters. Screening by yeast one-hybrid assays indicated a possible interaction between ZmbZIP22 and the promoters of eight key starch enzyme genes. Collectively, our results indicated that ZmbZIP22 functions as a negative regulator of starch synthesis, and suggest that this occurs through the regulation of key sugar and starch metabolism genes in maize.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Endosperma/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Amido/metabolismo , Zea mays/metabolismo , Amilose/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Southern Blotting , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Análise de Sequência de DNA , Transcriptoma , Técnicas do Sistema de Duplo-Híbrido , Zea mays/genética
12.
Curr Protoc Toxicol ; 80(1): e75, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30982231

RESUMO

A single cell can contain several thousand copies of the mitochondrial DNA genome or mtDNA. Tools for assessing mtDNA content are necessary for clinical and toxicological research, as mtDNA depletion is linked to genetic disease and drug toxicity. For instance, mtDNA depletion syndromes are typically fatal childhood disorders that are characterized by severe declines in mtDNA content in affected tissues. Mitochondrial toxicity and mtDNA depletion have also been reported in human immunodeficiency virus-infected patients treated with certain nucleoside reverse transcriptase inhibitors. Further, cell culture studies have demonstrated that exposure to oxidative stress stimulates mtDNA degradation. Here we outline a Southern blot and nonradioactive digoxigenin-labeled probe hybridization method to estimate mtDNA content in human genomic DNA samples. © 2019 by John Wiley & Sons, Inc.


Assuntos
Southern Blotting/métodos , Sondas de DNA/metabolismo , DNA Mitocondrial/genética , Células Cultivadas , Enzimas de Restrição do DNA/genética , Digoxigenina , Eletroforese em Gel de Ágar , Humanos , Plasmídeos/genética , Coloração e Rotulagem
13.
Methods Mol Biol ; 1942: 11-27, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900172

RESUMO

Fragile X syndrome (FXS) is characterized by mental retardation and in the vast majority of cases it is caused by expansion of CGG trinucleotide repeats in the 5' untranslated region (or UTR) in the FMR1 gene on the X chromosome. The size and methylation status of CGG repeats are correlated with the clinical phenotype of FMR1-related disorders. The methods used for clinical genetic testing of FXS include polymerase chain reaction (PCR) amplification and Southern blot analyses, which effectively detect alleles with repeats in the normal, intermediate, premutation, and full mutation size ranges, as well as the methylation status of FMR1 promoter region.


Assuntos
Southern Blotting/métodos , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/diagnóstico , Testes Genéticos , Mutação , Reação em Cadeia da Polimerase/métodos , Metilação de DNA , Feminino , Síndrome do Cromossomo X Frágil/genética , Humanos , Masculino , Repetições de Trinucleotídeos
14.
Indian J Tuberc ; 66(1): 92-98, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30797291

RESUMO

BACKGROUND: Tuberculosis (TB) caused 1.8 million deaths worldwide with increased multiple drug resistance (MDR) cases estimated 4.8 lakhs in the year 2015. ß-Lactam antibiotics could be a hope for TB treatment. Therefore, in this study, uniformity in the biochemical and molecular nature of ß-lactamases was analyzed to evaluate the potential of ß-lactam antibiotics as a treatment regimen against Mycobacterium tuberculosis (MTB). MATERIALS AND METHODS: ß-Lactamase enzymes in 233 MTB clinical isolates along with control H37Rv strain were characterized by enzyme kinetic using nitrocefin and cefotaxime as a substrate, isoelectric points by isoelectric focusing electrophoresis (IEF) and by PCR and Southern blotting. RESULTS: Enzyme kinetics showed Km and Vmax for nitrocefin in the range of 56-69µM and 7.00-11IU/lit respectively, for cefotaxime in the range of 0.35-0.59µM and 18-25IU/lit respectively. ß-Lactamase showed high affinity for clavulanic acid an inhibitor of Extended-Spectrum ß-lactamase enzymes (ESBLs). The pIs of 4.9 and 5.1 were observed for all the MTB clinical isolates and control H37Rv. Southern blotting confirmed the presence of blaC sequence in MTB chromosomal DNA. CONCLUSION: This confirmed that MTB ß-lactamase enzymes belong to the Class A, group 2be Extended Spectrum ß-Lactamases with no biochemical or molecular polymorphism. ESBLs are mainly responsible for resistance against ß-lactam antibiotics in MTB. Thus ESBLs could be the potential therapeutic target for TB treatment using ß-lactam antibiotics in combination with ß-lactamase inhibitors like sulbactam and sodium clavulanate.


Assuntos
Antibacterianos/metabolismo , Mycobacterium tuberculosis/metabolismo , beta-Lactamases/metabolismo , beta-Lactamas/metabolismo , Southern Blotting , Cefotaxima/metabolismo , Cefalosporinas/metabolismo , Ácido Clavulânico/metabolismo , Humanos , Focalização Isoelétrica , Cinética , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase , Resistência beta-Lactâmica/genética , beta-Lactamases/genética
15.
Invest Ophthalmol Vis Sci ; 60(2): 779-786, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30811544

RESUMO

Purpose: CTG trinucleotide repeat (TNR) expansion is frequently found in transcription factor 4 (TCF4) in Fuchs' endothelial corneal dystrophy (FECD), though the effect of TNR expansion on FECD pathophysiology remains unclear. The purpose of this study was to evaluate the effect of TNR expansion on TCF4 expression in corneal endothelium of patients with FECD. Methods: Peripheral blood DNA and Descemet membrane with corneal endothelium were obtained from 203 German patients with FECD. The CTG TNR repeat length in TCF4 was determined by short tandem repeat (STR) assays and Southern blotting using genomic DNA. Genotyping of rs613872 in TCF4 was performed by PCR. TCF4 mRNA levels in corneal endothelium were evaluated by quantitative PCR using three different probes. Control corneal endothelial samples were obtained from 35 non-FECD subjects. Results: The STR assay and Southern blotting showed that 162 of the 203 patients with FECD (80%) harbored CTG trinucleotide repeat lengths larger than 50. Quantitative PCR using all three probes demonstrated that TCF4 mRNA is significantly upregulated in the corneal endothelium of patients with FECD, regardless of the presence of TNR expansion. However, the length of the TNR tended to show a positive correlation with TCF4 expression level. No correlation was shown between the genotype of TCF4 SNP, rs613872, and the level of TCF4 expression. Conclusions: Our findings showed that TCF4 mRNA is upregulated in the corneal endothelium of patients with FECD. Further studies on the effects of TCF4 upregulation on corneal endothelial cell function will aid in understanding the pathophysiology of FECD.


Assuntos
Distrofia Endotelial de Fuchs/genética , Regulação da Expressão Gênica/fisiologia , RNA Mensageiro/genética , Fator de Transcrição 4/genética , Expansão das Repetições de Trinucleotídeos , Adulto , Idoso , Idoso de 80 Anos ou mais , Southern Blotting , Feminino , Técnicas de Genotipagem , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
16.
Nat Commun ; 10(1): 759, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770810

RESUMO

Mitochondrial DNA (mtDNA) deletions are associated with mitochondrial disease, and also accumulate during normal human ageing. The mechanisms underlying mtDNA deletions remain unknown although several models have been proposed. Here we use deep sequencing to characterize abundant mtDNA deletions in patients with mutations in mitochondrial DNA replication factors, and show that these have distinct directionality and repeat characteristics. Furthermore, we recreate the deletion formation process in vitro using only purified mitochondrial proteins and defined DNA templates. Based on our in vivo and in vitro findings, we conclude that mtDNA deletion formation involves copy-choice recombination during replication of the mtDNA light strand.


Assuntos
DNA Mitocondrial/genética , Deleção de Sequência/genética , Southern Blotting , Replicação do DNA/genética , Humanos , Proteínas Mitocondriais/genética , Mutação/genética
17.
Am J Hum Genet ; 104(1): 35-44, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30554721

RESUMO

Baratela-Scott syndrome (BSS) is a rare, autosomal-recessive disorder characterized by short stature, facial dysmorphisms, developmental delay, and skeletal dysplasia caused by pathogenic variants in XYLT1. We report clinical and molecular investigation of 10 families (12 individuals) with BSS. Standard sequencing methods identified biallelic pathogenic variants in XYLT1 in only two families. Of the remaining cohort, two probands had no variants and six probands had only a single variant, including four with a heterozygous 3.1 Mb 16p13 deletion encompassing XYLT1 and two with a heterozygous truncating variant. Bisulfite sequencing revealed aberrant hypermethylation in exon 1 of XYLT1, always in trans with the sequence variant or deletion when present; both alleles were methylated in those with no identified variant. Expression of the methylated XYLT1 allele was severely reduced in fibroblasts from two probands. Southern blot studies combined with repeat expansion analysis of genome sequence data showed that the hypermethylation is associated with expansion of a GGC repeat in the XYLT1 promoter region that is not present in the reference genome, confirming that BSS is a trinucleotide repeat expansion disorder. The hypermethylated allele accounts for 50% of disease alleles in our cohort and is not present in 130 control subjects. Our study highlights the importance of investigating non-sequence-based alterations, including epigenetic changes, to identify the missing heritability in genetic disorders.


Assuntos
Anormalidades Múltiplas/genética , Metilação de DNA/genética , Epigênese Genética/genética , Éxons/genética , Mutação , Pentosiltransferases/genética , Expansão das Repetições de Trinucleotídeos/genética , Alelos , Southern Blotting , Estudos de Coortes , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Linhagem , Sulfitos/metabolismo , Síndrome
18.
J Med Microbiol ; 68(2): 201-205, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30570481

RESUMO

Between September 2013 and March 2016, 26 (1.95 %) of 1333 Staphylococcus aureus clinical isolates from a Chinese hospital were found to be resistant to mupirocin, including 18 (1.35 %) with high-level mupirocin resistance and 8 (0.6 %) with low-level mupirocin resistance. Among the 18 isolates with high-level mupirocin resistance, 17 were associated with plasmid-mediated mupA. Meanwhile, the 8 isolates with low-level mupirocin resistance were shown to have a V588F mutation in ileS. A total of 14 sequence types (STs) and 18 spa types were identified. All four isolates with t062 belonged to ST965. Three ST5-MRSA-SCCmec II were linked to t311, which was not previously reported. Furthermore, ST764-MRSA-SCCmec II-t002, exclusively found in Japan before, was identified in this study. In conclusion, we observed relatively low prevalence of mupirocin resistance among S. aureus with considerable heterogeneity in East China. Newly emerging MRSA clones with high-level mupirocin resistance should be of concern.


Assuntos
Antibacterianos/farmacologia , Mupirocina/farmacologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Southern Blotting , China , Contagem de Colônia Microbiana , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Farmacorresistência Bacteriana , Eletroforese em Gel de Campo Pulsado , Humanos , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Tipagem de Sequências Multilocus , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Mutação Puntual , Reação em Cadeia da Polimerase , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Centros de Atenção Terciária
19.
Methods Mol Biol ; 1902: 123-136, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30543066

RESUMO

In this chapter, we present detailed experimental procedures for investigating integration patterns of transgenes in cotton genome. We use conventional PCR and genomic Southern blot hybridization to characterize integration of T-DNA components and vector backbone fragments. For multiple-copy insertions into the same site (complex loci), transgene/transgene junctions (including canonical and truncated T-DNA and transgene involved vector backbone sequences) are characterized by PCR and sequencing. Inverse PCR and sequencing are used to characterize transgene/cotton genome junctions. Distribution of T-DNA insertion in cotton genome is evaluated by analysis of transgene flanking sequences. The pre-insertion sites can also be cloned and sequenced (based on the flanking sequences) for survey of genomic structure changes brought by transgene integration by comparing a pre-insertion site with corresponding transgene/plant junctions.


Assuntos
Genoma de Planta , Genômica , Gossypium/genética , Transgenes , Southern Blotting , Vetores Genéticos/genética , Genômica/métodos , Plantas Geneticamente Modificadas , Análise de Sequência de DNA , Transformação Genética
20.
Plant Cell Environ ; 42(4): 1112-1124, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30311663

RESUMO

Sedum plumbizincicola is able to hyperaccumulate cadmium (Cd), a nonessential and highly toxic metal, in the above-ground tissues, but the mechanisms for its Cd hypertolerance are not fully understood. Here, we show that the heavy metal ATPase 1 (SpHMA1) of S. plumbizincicola plays an important role in chloroplast Cd detoxification. Compared with the HMA1 ortholog in the Cd nonhyperaccumulating ecotype of Sedum alfredii, the expression of SpHMA1 in the leaves of S. plumbizincicola was >200 times higher. Heterologous expression of SpHMA1 in Saccharomyces cerevisiae increased Cd sensitivity and Cd transport activity in the yeast cells. The SpHMA1 protein was localized to the chloroplast envelope. SpHMA1 RNA interference transgenic plants and CRISPR/Cas9-induced mutant lines showed significantly increased Cd accumulation in the chloroplasts compared with wild-type plants. Chlorophyll fluorescence imaging analysis revealed that the photosystem II of SpHMA1 knockdown and knockout lines suffered from a much higher degree of Cd toxicity than wild type. Taken together, these results suggest that SpHMA1 functions as a chloroplast Cd exporter and protects photosynthesis by preventing Cd accumulation in the chloroplast in S. plumbizincicola and hyperexpression of SpHMA1 is an important component contributing to Cd hypertolerance in S. plumbizincicola.


Assuntos
Adenosina Trifosfatases/metabolismo , Cádmio/metabolismo , Cloroplastos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismo , Sedum/metabolismo , Southern Blotting , Organismos Geneticamente Modificados , Fotossíntese , Folhas de Planta/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sedum/fisiologia
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