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1.
N Engl J Med ; 381(17): 1644-1652, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31597037

RESUMO

Genome sequencing is often pivotal in the diagnosis of rare diseases, but many of these conditions lack specific treatments. We describe how molecular diagnosis of a rare, fatal neurodegenerative condition led to the rational design, testing, and manufacture of milasen, a splice-modulating antisense oligonucleotide drug tailored to a particular patient. Proof-of-concept experiments in cell lines from the patient served as the basis for launching an "N-of-1" study of milasen within 1 year after first contact with the patient. There were no serious adverse events, and treatment was associated with objective reduction in seizures (determined by electroencephalography and parental reporting). This study offers a possible template for the rapid development of patient-customized treatments. (Funded by Mila's Miracle Foundation and others.).


Assuntos
Proteínas de Membrana Transportadoras/genética , Mutagênese Insercional , Lipofuscinoses Ceroides Neuronais/tratamento farmacológico , Lipofuscinoses Ceroides Neuronais/genética , Oligonucleotídeos Antissenso/uso terapêutico , Medicina de Precisão , Doenças Raras/tratamento farmacológico , Biópsia , Criança , Desenvolvimento Infantil , Descoberta de Drogas , Drogas em Investigação/uso terapêutico , Eletroencefalografia , Feminino , Humanos , Testes Neuropsicológicos , RNA Mensageiro , Convulsões/diagnóstico , Convulsões/tratamento farmacológico , Pele/patologia , Sequenciamento Completo do Genoma
2.
Gene ; 720: 144094, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31476407

RESUMO

Fourteen different insertion sequences belonging to seven families were identified in the genome of Streptococcus agalactiae. Among them, IS1548, a mobile element of the ISAs1 family, was linked to clonal complex (CC) 19 strains associated with neonatal meningitis and endocarditis. IS1548 impacts S. agalactiae in two reported ways: i) inactivation of virulence genes by insertion in an open reading frame (e.g. hylB or cpsD), ii) positive modulation of the expression of a downstream gene by insertion in an intergenic region (e.g. lmb). We previously identified an unknown integration site of IS1548 in the intergenic region between the folK and the murB genes involved in folate and peptidoglycan biosynthesis, respectively. In this work, we analyzed the prevalence of IS1548 in a large collection of nine hundred and eleven S. agalactiae strains. IS1548 positive strains belong to twenty-nine different sequence types and to ten CCs. The majority of them were, however, clustered within sequence type 19 and sequence type 22, belonging to CC19 and CC22, respectively. In contrast, IS1548 targets the folK-murB intergenic region exclusively in CC19 strains. We evaluated the impact of the insertion of IS1548 on the expression of murB by locating transcriptional promoters influencing its expression in the presence or absence of IS1548 and by comparative ß-galactosidase transcriptional fusion assays. We found that in the absence of IS1548, genes involved in folate biosynthesis are co-transcribed with murB. As it was postulated that a folic acid mediated reaction may be involved in cell wall synthesis, this co-transcription could be necessary to synchronize these two processes. The insertion of IS1548 in the folK-murB intergenic region disrupt this co-transcription. Interestingly, we located a promoter at the right end of IS1548 that is able to initiate additional transcripts of murB. The insertion of IS1548 in this region has thus a dual and divergent impact on the expression of murB. By comparative ß-galactosidase transcriptional fusion assays, we showed that, consequently, the overall impact of the insertion of IS1548 results in a minor decrease of murB gene transcription. This study provides new insights into gene expression effects mediated by IS1548 in S. agalactiae.


Assuntos
Proteínas de Bactérias/genética , DNA Intergênico , Regulação Bacteriana da Expressão Gênica , Sequências Repetitivas Dispersas , Mutagênese Insercional , Peptidoglicano/biossíntese , Streptococcus agalactiae/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/genética , Regiões Promotoras Genéticas , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/crescimento & desenvolvimento , Streptococcus agalactiae/metabolismo
3.
Microbiol Res ; 228: 126306, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31422233

RESUMO

The mariner transposon family of Himar1 has been widely used for the random mutagenesis of bacteria to generate single insertions into the chromosome. Here, a versatile toolbox of mariner transposon derivatives was generated and applied to the functional genomics investigation of fish pathogen Edwardsiella piscicida. In this study, we combined the merits of the random mutagenesis of mariner transposon and common efficient reporter marker genes or regulatory elements, mCherry, gfp, luxAB, lacZ, sacBR, and PBAD and antibiotic resistance cassettes to construct a series of derivative transposon vectors, pMmch, pMKGR, pMCGR, pMXKGR, pMLKGR, pMSGR, and pMPR, based on the initial transposon pMar2xT7. The function and effectiveness of the modified transposons were verified by introducing them into E. piscicida EIB202. Based on the toolbox, a transposon insertion mutant library containing approximately 3.0 × 105 distinct mutants was constructed to explore the upstream regulators of esrB, the master regulator of the type III and type VI secretion systems (T3/T6SS) in E. piscicida. Following analysis by Con-ARTIST, ETAE_3474, annotated as fabR and involved in fatty acid metabolism, was screened out and identified as a novel regulator mediating T3SS and T6SS expression. In addition, the fabR mutants displayed critical virulence attenuation in turbot. Due to the broad-range host compatibility of mariner transposons, the newly built transposon toolbox can be applied for functional genomics studies in various bacteria.


Assuntos
Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Edwardsiella/genética , Regulação Bacteriana da Expressão Gênica/genética , Testes Genéticos/métodos , Genoma Bacteriano/genética , Animais , Mapeamento Cromossômico , Farmacorresistência Bacteriana/genética , Ácidos Graxos/metabolismo , Doenças dos Peixes/microbiologia , Biblioteca Gênica , Genes Reporter/genética , Genômica/métodos , Mutagênese Insercional/métodos , Fatores de Transcrição/genética , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo VI/genética , Virulência , Fatores de Virulência/genética
4.
Neuron ; 103(4): 583-597.e8, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31272828

RESUMO

Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the "plug-and-play" nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.


Assuntos
Técnicas de Introdução de Genes/métodos , Genômica/métodos , Engenharia de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Dependovirus/genética , Edição de Genes/métodos , Vetores Genéticos/genética , Humanos , Imunoquímica/métodos , Inteínas , Camundongos , Mutagênese Insercional , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteômica , RNA Guia/genética , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência do Ácido Nucleico
5.
Pan Afr Med J ; 32: 197, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31312309

RESUMO

Introduction: Just recently, it has been established that the angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism is linked to the pathogenesis and to the evolution of human cancers. Therefore, the present study was concerned with the investigation of an eventual association between glioma and I/D polymorphism of the ACE gene. Methods: The expression of ACE gene was detected by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis in 36 Algerian patients with glioma and 195 healthy controls. Results: In glioma cases, allelic frequencies and genotypes distribution of the ACE I/D polymorphism were different from controls cases. ACE DD genotype were highly presented in glioma cases (63.9%) than controls (33.8%) and conferred 3.64-fold risk for predisposition in glioma cases (vs ID genotype, p<0.001). Recessive model (ACE II + ID genotypes vs DD) was associated with a 72% reduced risk of glioma (OR = 0.28, 95% CI: 0.13-0.60, p <0.001). Per copy D allele frequency was found higher in glioma cases (79.2%) than in controls (63.3 %), OR = 2.20, 95% CI: 1.20 - 4.03, p = 0.009. Conclusion: The obtained data showed that the presence of the D allele might be a risk factor for the development of glioma. Further studies considering different ethnic groups with large samples are required to confirm this finding.


Assuntos
Neoplasias Encefálicas/genética , Predisposição Genética para Doença , Glioma/genética , Peptidil Dipeptidase A/genética , Adulto , Argélia , Estudos de Casos e Controles , Feminino , Deleção de Genes , Frequência do Gene , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutagênese Insercional , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Fatores de Risco
6.
Plant Mol Biol ; 101(1-2): 183-202, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31286324

RESUMO

KEY MESSAGE: Isoforms of 2-OGDH E1 subunit are not functionally redundant in plant growth and development of A. thaliana. The tricarboxylic acid cycle enzyme 2-oxoglutarate dehydrogenase (2-OGDH) converts 2-oxoglutarate (2-OG) to succinyl-CoA concomitant with the reduction of NAD+. 2-OGDH has an essential role in plant metabolism, being both a limiting step during mitochondrial respiration as well as a key player in carbon-nitrogen interactions. In Arabidopsis thaliana two genes encode for E1 subunit of 2-OGDH but the physiological roles of each isoform remain unknown. Thus, in the present study we isolated Arabidopsis T-DNA insertion knockout mutant lines for each of the genes encoding the E1 subunit of 2-OGDH enzyme. All mutant plants exhibited substantial reduction in both respiration and CO2 assimilation rates. Furthermore, mutant lines exhibited reduced levels of chlorophylls and nitrate, increased levels of sucrose, malate and fumarate and minor changes in total protein and starch levels in leaves. Despite the similar metabolic phenotypes for the two E1 isoforms the reduction in the expression of each gene culminated in different responses in terms of plant growth and seed production indicating distinct roles for each isoform. Collectively, our results demonstrated the importance of the E1 subunit of 2-OGDH in both autotrophic and heterotrophic tissues and suggest that the two E1 isoforms are not functionally redundant in terms of plant growth in A. thaliana.


Assuntos
Arabidopsis/enzimologia , Carbono/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Nitrogênio/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Complexo Cetoglutarato Desidrogenase/genética , Mitocôndrias/enzimologia , Mutagênese Insercional , Nitratos/metabolismo , Fenótipo , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Isoformas de Proteínas , Subunidades Proteicas , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento
7.
Nature ; 571(7764): 219-225, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31189177

RESUMO

Conventional CRISPR-Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a notable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR-Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.


Assuntos
Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Edição de Genes/métodos , Mutagênese Insercional/métodos , RNA Bacteriano/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Genoma Bacteriano/genética , Integrases/genética , Integrases/metabolismo , Mutagênese Sítio-Dirigida/métodos , RNA Guia/genética , Especificidade por Substrato , Vibrio cholerae/genética
9.
PLoS Genet ; 15(6): e1008168, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199785

RESUMO

The lack of predictive preclinical models is a fundamental barrier to translating knowledge about the molecular pathogenesis of cancer into improved therapies. Insertional mutagenesis (IM) in mice is a robust strategy for generating malignancies that recapitulate the extensive inter- and intra-tumoral genetic heterogeneity found in advanced human cancers. While the central role of "driver" viral insertions in IM models that aberrantly increase the expression of proto-oncogenes or disrupt tumor suppressors has been appreciated for many years, the contributions of cooperating somatic mutations and large chromosomal alterations to tumorigenesis are largely unknown. Integrated genomic studies of T lineage acute lymphoblastic leukemias (T-ALLs) generated by IM in wild-type (WT) and Kras mutant mice reveal frequent point mutations and other recurrent non-insertional genetic alterations that also occur in human T-ALL. These somatic mutations are sensitive and specific markers for defining clonal dynamics and identifying candidate resistance mechanisms in leukemias that relapse after an initial therapeutic response. Primary cancers initiated by IM and resistant clones that emerge during in vivo treatment close key gaps in existing preclinical models, and are robust platforms for investigating the efficacy of new therapies and for elucidating how drug exposure shapes tumor evolution and patterns of resistance.


Assuntos
Genômica , Leucemia-Linfoma Linfoblástico de Células T Precursoras/dietoterapia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Linhagem Celular Tumoral , Aberrações Cromossômicas , Evolução Clonal/genética , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Camundongos , Mutagênese Insercional/genética , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia
10.
Am J Vet Res ; 80(7): 680-688, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31246118

RESUMO

OBJECTIVE: To examine effects of a common mutation (2-base insertion in exon 5) of the TP53 gene on biological function of p53 protein in canine histiocytic sarcoma cells. SAMPLE: Canine histiocytic tumor cell lines DH82 with deletion of TP53 and CHS-3 with the wild-type TP53 and canine wild-type and mutant TP53 fragments. PROCEDURES: Wild-type or mutant TP53 with a polyprotein peptide tag at the N-terminus was transduced into DH82 and CHS-3 cells. Expression of p53 protein, changes in function as a transcription factor, and susceptibility to doxorubicin and nimustine were compared. RESULTS: Transduced p53 protein was detected in wild-type TP53-transduced DH82 and CHS-3 cells, whereas expression was not detected in mutant TP53-transduced cells. There were significant increases in expression of target genes of p53 protein, including p21 and MDM2, in wild-type TP53-transduced cells, compared with results for native and mock-transfected cells, but not in mutant TP53-transduced cells. There was no significant difference in drug susceptibilities among native and derivative cells of CHS-3. However, cell viabilities of wild-type TP53-transduced DH82 cells incubated with doxorubicin were significantly lower than viabilities of native, mock-transfected, and AT insertion mutation-TP53-transduced DH82 cells; susceptibility to nimustine did not differ significantly among cells. CONCLUSIONS AND CLINICAL RELEVANCE: Expression of p53 protein and its function as a transcription factor were lost after addition of a 2-base insertion in the TP53 gene in canine histiocytic tumor cells. Additional studies are needed to investigate the clinical relevance of this mutation in histiocytic sarcomas of dogs.


Assuntos
Cães , Mutagênese Insercional/fisiologia , Proteína Supressora de Tumor p53/genética , Animais , Linhagem Celular Tumoral , Doenças do Cão/fisiopatologia , Genes p53/genética , Sarcoma Histiocítico/fisiopatologia , Sarcoma Histiocítico/veterinária , Mutação , Proteína Supressora de Tumor p53/metabolismo
11.
Nat Commun ; 10(1): 2866, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253785

RESUMO

Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPR-Cas9 by homology-dependent repair (HDR) is limited by the competing error-prone non-homologous end-joining (NHEJ) DNA repair pathway. Here, we define a safer and efficient system that promotes HDR-based precise genome editing, while reducing NHEJ locally, only at CRISPR-Cas9-induced DSBs. We fused a dominant-negative mutant of 53BP1, DN1S, to Cas9 nucleases, and the resulting Cas9-DN1S fusion proteins significantly block NHEJ events specifically at Cas9 cut sites and improve HDR frequency; HDR frequency reached 86% in K562 cells. Cas9-DN1S protein maintains this effect in different human cell types, including leukocyte adhesion deficiency (LAD) patient-derived immortalized B lymphocytes, where nearly 70% of alleles were repaired by HDR and 7% by NHEJ. Our CRISPR-Cas9-DN1S system is clinically relevant to improve the efficiencies of precise gene correction/insertion, significantly reducing error-prone NHEJ events at the nuclease cleavage site, while avoiding the unwanted effects of global NHEJ inhibition.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Reparo do DNA , Edição de Genes/métodos , Reparo de DNA por Recombinação/fisiologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Humanos , Mutagênese Insercional , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
12.
Anticancer Res ; 39(6): 2757-2765, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31177111

RESUMO

BACKGROUND/AIM: Clear cell renal cell carcinoma (ccRCC) shows variable chromosomal abnormalities. The aim of this study was to assess the prognostic role of ccRCC chromosomal abnormalities in a single-center cohort with an extended follow-up. MATERIALS AND METHODS: A systematic cytogenetic analysis was performed in 283 consecutive surgically-treated patients for renal masses between 1997 and 2002. Kaplan-Meier and multivariable Cox regression (MCR) models were used to calculate cancer specific survival (CSS). RESULTS: Among 174 ccRCC patients, the most common abnormality was deletion in chromosome 3 (54.6%). At a median follow-up of 119 months, 38 patients (21.8%) died from RCC. At MCR models, worse CSS was independently predicted by deletions in chromosomes 2, 19, 20 or 22 and insertions in chromosome 18. CONCLUSION: Specific ccRCC chromosomal abnormalities are independently associated with worse CSS. Cytogenetic evaluation may direct further genetic analysis for personalized prognostic stratification.


Assuntos
Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/cirurgia , Aberrações Cromossômicas , Cromossomos Humanos/genética , Neoplasias Renais/mortalidade , Neoplasias Renais/cirurgia , Idoso , Carcinoma de Células Renais/genética , Deleção Cromossômica , Citogenética , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/genética , Masculino , Pessoa de Meia-Idade , Mutagênese Insercional , Prognóstico , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Resultado do Tratamento
13.
Mol Plant Microbe Interact ; 32(9): 1063-1066, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30958087

RESUMO

The pRC system is an efficient tool for genetic studies in Ralstonia solanacearum, ensuring direct insertion of foreign gene elements into Ralstonia chromosome downstream of glms. This system is designed for double recombination across glms and the downstream region, which considerably simplifies genetic studies of complementation, overexpression, pathogenicity, and in-vivo promoter activity assays with monocopy in R. solanacearum, one of the most destructive plant-pathogenic bacteria worldwide. R. solanacearum is extremely heterogeneous and is currently referred to as a Ralstonia solanacearum species complex (RSSC). The glms gene is greatly conserved, but its downstream regions are mostly different in the RSSC, which limits the application of the current pRC plasmid in the RSSC. We compared all existing 132 genome sequences in a precise genomic glms downstream region and confirmed that the pRC system is appropriate for application of chromosomal integration in all RSSC strains but needs respective reconstruction on current pRC plasmids, since glms downstream regions are greatly variable in the RSSC. RSSC strains can be grouped according to identical glms downstream regions. This grouping provides valuable information for gene insertion in this chromosomal region, as it facilitates universal application of the pRC system in RSSC strains.


Assuntos
Mutagênese Insercional , Plasmídeos , Ralstonia solanacearum/genética , Cromossomos/genética , Genoma de Planta/genética , Plasmídeos/genética , Virulência/genética
14.
Methods Mol Biol ; 1968: 63-78, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929206

RESUMO

The ability of Streptococcus pneumoniae (the pneumococcus) to transform is particularly convenient for genome engineering. Several protocols relying on sequential positive and negative selection strategies have been described to create directed markerless modifications, including deletions, insertions, or point mutations. Transformation with DNA fragments carrying long flanking homology sequences is also used to generate mutations without selection but it requires high transformability. Here, we present an optimized version of this method. As an example, we construct a strain harboring a translational fusion ftsZ-mTurquoise at the ftsZ locus. We provide instructions to produce a linear DNA fragment containing the chimeric construction and give details of the conditions to obtain optimal pneumococcal transformation efficiencies.


Assuntos
Cromossomos Bacterianos/genética , DNA Bacteriano/genética , Streptococcus pneumoniae/genética , Mutagênese Insercional , Mutação/genética , Recombinação Genética/genética
15.
Appl Microbiol Biotechnol ; 103(11): 4429-4441, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30972461

RESUMO

Biosurfactants are amphiphilic molecules that interact with the surfaces of liquids leading to many useful applications. Most biosurfactants have been identified from cultured microbial sources, leaving a largely untapped resource of uncultured bacteria with potentially novel biosurfactant structures. To access the uncultured bacteria, a metagenomic library was constructed in Escherichia coli from environmental DNA within an E. coli, Pseudomonas putida and Streptomyces lividans shuttle vector. Phenotypic screening of the library in E. coli and P. putida by the paraffin spray assay identified a P. putida clone with biosurfactant activity. Sequence analysis and transposon mutagenesis confirmed that an ornithine acyl-ACP N-acyltransferase was responsible for the activity. Although the fosmid was not active in E. coli, overexpression of the olsB gene could be achieved under the control of the inducible T7 promoter, resulting in lyso-ornithine lipid production and biosurfactant activity in the culture supernatants. Screening for activity in more than one host increases the range of sequences that can be identified through metagenomic, since olsB would not have been identified if only E. coli had been used as a host. The potential of lyso-ornithine lipids as a biosurfactant has not been fully explored. Here, we present several biosurfactant parameters of lyso-ornithine lipid to assess its suitability for industrial application.


Assuntos
Acetiltransferases/metabolismo , Metagenômica/métodos , Ornitina/análogos & derivados , Tensoativos/metabolismo , Acetiltransferases/genética , Elementos de DNA Transponíveis , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca Gênica , Testes Genéticos , Vetores Genéticos , Lipídeos , Mutagênese Insercional , Ornitina/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Análise de Sequência de DNA
16.
Appl Microbiol Biotechnol ; 103(11): 4549-4564, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31001742

RESUMO

Previously, we have developed an L-threonine-producing Escherichia coli strain TWF006 in which the regulator-encoding gene iclR was deleted. In this study, further modifications were performed on TWF006 to increase L-threonine yield. Firstly, the regulator-encoding gene fadR was deleted in TWF006, and the resulting strain TWF031 produced 18.86 g L-threonine from 30 g glucose after 24-h cultivation. Secondly, the regulator-encoding genes fabR and lacI in TWF031 were deleted, and the resulting strain TWF033 produced 19.21 g L-threonine from 30 g glucose after 24-h cultivation. Thirdly, additional copies of aceBA and fadBA were inserted into the lacZ locus of TWF033 and the native promoter of acs was replaced by the Ptac-trc; the resulting strain TWF038 produced 20.3 g L-threonine from 30 g glucose after 24-h cultivation. Finally, the genes ppnK, thrA*BC-rhtC, aspC, and ppc were inserted into the chromosome of TWF038; the resulting strain TWF044 produced 21.64 g L-threonine from 30 g glucose, or 28.49 g L-threonine from 40 g glucose after 24-h cultivation. After 48-h fed-batch fermentation, TWF044 produced 103.89 g/l L-threonine. The results suggest that coupling the fatty acid degradation and L-threonine biosynthesis pathway via the glyoxylate shunt could efficiently increase L-threonine production in E. coli.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Engenharia Metabólica/métodos , Proteínas Repressoras/genética , Treonina/biossíntese , Fatores de Transcrição/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fermentação , Glucose/metabolismo , Mutagênese Insercional , Recombinação Genética
17.
BMC Res Notes ; 12(1): 222, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975199

RESUMO

OBJECTIVE: Alu elements are retroposons that invaded the primate genome and shaped its biology. Some Alus inserted recently and are polymorphic in the human population. It is these Alus that are being sought after in disease association studies and regulatory biology. Discovering polymorphic Alus in the human genome can open areas of new research in these fields. RESULTS: Using the polymerase chain reaction on genomic DNA, we identified a polymorphic Alu in the flanking region of the TFAP2B and TFAP2D genes. The new insert was found in higher frequency in Europeans (0.4) and Asians (0.38) and lower frequency in Africans (0.25). We also show this Alu to be part of a 3 Alu cassette that is human specific. The TFAP2B and TFAP2D genes encode members of the transcription factor AP-2, which plays a role in organ development. The insertion of this Alu cassette flanking the transcription factor genes distinguishes humans from the primates. This cassette can possibly affect the regulation of both genes or alternately provoke genomic deletions, which we have shown in this study. Its presence in such a location is intriguing and unquestionably opens an investigational window in disease association studies and in the field of gene regulation.


Assuntos
Região 3'-Flanqueadora , Região 5'-Flanqueadora , Elementos Alu , Genoma Humano , Mutagênese Insercional , Fator de Transcrição AP-2/genética , Grupo com Ancestrais do Continente Africano , Grupo com Ancestrais do Continente Asiático , Sequência de Bases , Bases de Dados de Ácidos Nucleicos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Grupo com Ancestrais do Continente Europeu , Expressão Gênica , Humanos , Polimorfismo Genético , Cultura Primária de Células , Alinhamento de Sequência
18.
Gene ; 707: 22-29, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31026568

RESUMO

Reinforcing the immunity of pregnant sows can not only improve their own health condition but also increase the survival rate and healthy status of their piglets. This study aims to find single-nucleotide polymorphism (SNP) and molecular markers that are associated with the immune traits of pregnant sows. SLA-DOB and CD4 were selected as candidate genes, and blood samples were randomly collected from pregnant Landrace sows and used to detect T-lymphocyte subsets, interferon alpha, interleukin 6, Toll-like receptor 3, serum antibody immunoglobulin G, and porcine reproductive and respiratory syndrome virus-specific antibody. Then, association analyses were conducted for the polymorphic sites of candidate genes with immune traits. We found 12 mutations in the two genes and conducted an association study with eight of them. Our results indicated that among the eight mutations, SNP1, SNP2, and SNP3 of the SLA-DOB gene and Ins9, SNP10, and SNP11 in the CD4 gene are newly discovered mutations. Except for SNP1, SNP3, and SNP11, the other five SNPs are associated with at least one immune trait tested. Especially, SNP2 and Ins9 are significantly associated with at least one of the T-lymphocyte subgroups and at least one antibody. These novel mutations have potential important effects on the polymorphic loci of the above immune traits in pregnant sows. The results suggest that the SLA-DOB and CD4 genes and their genetic mutations can be considered as important candidate genes and mutations for the immunity of pregnant sows.


Assuntos
Antígenos CD4/genética , Estudos de Associação Genética/veterinária , Antígenos de Histocompatibilidade Classe I/genética , Imunidade , Mutação , Animais , Feminino , Haplótipos , Imunoglobulina G/sangue , Interleucina-6/sangue , Família Multigênica , Mutagênese Insercional , Polimorfismo de Nucleotídeo Único , Gravidez , Locos de Características Quantitativas , Suínos , Linfócitos T/imunologia , Receptor 3 Toll-Like/sangue , Fator de Necrose Tumoral alfa/sangue
19.
Mol Biol Evol ; 36(5): 974-989, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30938771

RESUMO

Because of their symbiotic origin, many mitochondrial proteins are well conserved across eukaryotic kingdoms. It is however less obvious how specific lineages have obtained novel nuclear-encoded mitochondrial proteins. Here, we report a case of mitochondrial neofunctionalization in plants. Phylogenetic analysis of genes containing the Domain of Unknown Function 295 (DUF295) revealed that the domain likely originated in Angiosperms. The C-terminal DUF295 domain is usually accompanied by an N-terminal F-box domain, involved in ubiquitin ligation via binding with ASK1/SKP1-type proteins. Due to gene duplication, the gene family has expanded rapidly, with 94 DUF295-related genes in Arabidopsis thaliana alone. Two DUF295 family subgroups have uniquely evolved and quickly expanded within Brassicaceae. One of these subgroups has completely lost the F-box, but instead obtained strongly predicted mitochondrial targeting peptides. We show that several representatives of this DUF295 Organellar group are effectively targeted to plant mitochondria and chloroplasts. Furthermore, many DUF295 Organellar genes are induced by mitochondrial dysfunction, whereas F-Box DUF295 genes are not. In agreement, several Brassicaceae-specific DUF295 Organellar genes were incorporated in the evolutionary much older ANAC017-dependent mitochondrial retrograde signaling pathway. Finally, a representative set of DUF295 T-DNA insertion mutants was created. No obvious aberrant phenotypes during normal growth and mitochondrial dysfunction were observed, most likely due to the large extent of gene duplication and redundancy. Overall, this study provides insight into how novel mitochondrial proteins can be created via "intercompartmental" gene duplication events. Moreover, our analysis shows that these newly evolved genes can then be specifically integrated into relevant, pre-existing coexpression networks.


Assuntos
Arabidopsis/genética , Duplicação Gênica , Proteínas Mitocondriais/genética , Família Multigênica , Análise Mutacional de DNA , DNA Bacteriano , Proteínas F-Box/genética , Expressão Gênica , Genoma de Planta , Mutagênese Insercional , Proteínas de Plantas/genética , Transdução de Sinais
20.
Nat Commun ; 10(1): 1939, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028275

RESUMO

Accurate and fast aligners are required to handle the steadily increasing volume of sequencing data. Here we present an approach allowing performant alignments of short reads (Illumina) as well as long reads (Pacific Bioscience, Ultralong Oxford Nanopore), while achieving high accuracy, based on a universal three-stage scheme. It is also suitable for the discovery of insertions and deletions that originate from structural variants. We comprehensively compare our approach to other state-of-the-art aligners in order to confirm its performance with respect to accuracy and runtime. As part of our algorithmic scheme, we introduce two line sweep-based techniques called "strip of consideration" and "seed harmonization". These techniques represent a replacement for chaining and do not rely on any specially tailored data structures. Additionally, we propose a refined form of seeding on the foundation of the FMD-index.


Assuntos
Algoritmos , DNA/química , Análise de Sequência de DNA/estatística & dados numéricos , Software , Sequência de Bases , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutagênese Insercional , Alinhamento de Sequência , Análise de Sequência de DNA/métodos , Deleção de Sequência , Homologia de Sequência do Ácido Nucleico
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