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1.
Nat Commun ; 11(1): 4046, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792488

RESUMO

2-oxoglutarate (2-OG or α-ketoglutarate) relates mitochondrial metabolism to cell function by modulating the activity of 2-OG dependent dioxygenases involved in the hypoxia response and DNA/histone modifications. However, metabolic pathways that regulate these oxygen and 2-OG sensitive enzymes remain poorly understood. Here, using CRISPR Cas9 genome-wide mutagenesis to screen for genetic determinants of 2-OG levels, we uncover a redox sensitive mitochondrial lipoylation pathway, dependent on the mitochondrial hydrolase ABHD11, that signals changes in mitochondrial 2-OG metabolism to 2-OG dependent dioxygenase function. ABHD11 loss or inhibition drives a rapid increase in 2-OG levels by impairing lipoylation of the 2-OG dehydrogenase complex (OGDHc)-the rate limiting step for mitochondrial 2-OG metabolism. Rather than facilitating lipoate conjugation, ABHD11 associates with the OGDHc and maintains catalytic activity of lipoyl domain by preventing the formation of lipoyl adducts, highlighting ABHD11 as a regulator of functional lipoylation and 2-OG metabolism.


Assuntos
Complexo Cetoglutarato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mitocôndrias/metabolismo , Mutagênese/fisiologia , Serina Proteases/metabolismo , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Células HeLa , Humanos , Complexo Cetoglutarato Desidrogenase/genética , Modelos Biológicos , Mutagênese/genética , Serina Proteases/genética
2.
Nat Commun ; 11(1): 4132, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807781

RESUMO

Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Guide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design.


Assuntos
Edição de Genes/métodos , RNA Guia/metabolismo , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Humanos , Mutagênese/genética , Mutagênese/fisiologia , RNA Guia/genética
3.
Nat Commun ; 11(1): 3469, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651386

RESUMO

Insertions and deletions (InDels) are frequently observed in natural protein evolution, yet their potential remains untapped in laboratory evolution. Here we introduce a transposon-based mutagenesis approach (TRIAD) to generate libraries of random variants with short in-frame InDels, and screen TRIAD libraries to evolve a promiscuous arylesterase activity in a phosphotriesterase. The evolution exhibits features that differ from previous point mutagenesis campaigns: while the average activity of TRIAD variants is more compromised, a larger proportion has successfully adapted for the activity. Different functional profiles emerge: (i) both strong and weak trade-off between activities are observed; (ii) trade-off is more severe (20- to 35-fold increased kcat/KM in arylesterase with 60-400-fold decreases in phosphotriesterase activity) and (iii) improvements are present in kcat rather than just in KM, suggesting adaptive solutions. These distinct features make TRIAD an alternative to widely used point mutagenesis, accessing functional innovations and traversing unexplored fitness landscape regions.


Assuntos
Mutação INDEL/genética , Evolução Molecular , Humanos , Mutagênese/genética , Mutagênese/fisiologia , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Biologia Sintética/métodos
4.
Mol Cell ; 78(6): 1166-1177.e6, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32497495

RESUMO

Human tumors with exonuclease domain mutations in the gene encoding DNA polymerase ε (POLE) have incredibly high mutation burdens. These errors arise in four unique mutation signatures occurring in different relative amounts, the etiologies of which remain poorly understood. We used CRISPR-Cas9 to engineer human cell lines expressing POLE tumor variants, with and without mismatch repair (MMR). Whole-exome sequencing of these cells after defined numbers of population doublings permitted analysis of nascent mutation accumulation. Unlike an exonuclease active site mutant that we previously characterized, POLE cancer mutants readily drive signature mutagenesis in the presence of functional MMR. Comparison of cell line and human patient data suggests that the relative abundance of mutation signatures partitions POLE tumors into distinct subgroups dependent on the nature of the POLE allele, its expression level, and MMR status. These results suggest that different POLE mutants have previously unappreciated differences in replication fidelity and mutagenesis.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Alelos , Linhagem Celular Tumoral , Reparo de Erro de Pareamento de DNA/fisiologia , Humanos , Mutagênese/genética , Mutação/genética , Neoplasias/genética , Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo
5.
Mutat Res ; 853: 503195, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32522347

RESUMO

Recent years have witnessed an expansion of mutagenesis research focusing on experimentally modeled genome-scale mutational signatures of carcinogens and of endogenous processes. Experimental mutational signatures can explain etiologic links to patterns found in human tumors that may be linked to same exposures, and can serve as biomarkers of exposure history and may even provide insights on causality. A number of innovative exposure models have been employed and reported, based on cells cultured in monolayers or in 3-D, on organoids, induced pluripotent stem cells, non-mammalian organisms, microorganisms and rodent bioassays. Here we discuss some of the latest developments and pros and cons of these experimental systems used in mutational signature analysis. Integrative designs that bring together multiple exposure systems (in vitro, in vivo and in silico pan-cancer data mining) started emerging as powerful tools to identify robust mutational signatures of the tested cancer risk agents. We further propose that devising a new generation of cell-based models is warranted to streamline systematic testing of carcinogen effects on the cell genomes, while seeking to increasingly supplant animal with non-animal systems to address relevant ethical issues and accentuate the 3R principles. We conclude that the knowledge accumulating from the growing body of signature modelling investigations has considerable power to advance cancer etiology studies and to support cancer prevention efforts through streamlined characterization of cancer-causing agents and the recognition of their specific effects.


Assuntos
Carcinógenos/toxicidade , Mutagênese/efeitos dos fármacos , Mutação/efeitos dos fármacos , Neoplasias/induzido quimicamente , Animais , Análise Mutacional de DNA/métodos , Genoma Humano/efeitos dos fármacos , Genoma Humano/genética , Humanos , Mutagênese/genética , Mutação/genética
6.
J Vis Exp ; (160)2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32597859

RESUMO

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. The non-replicative elementary body (EB) initiates infection of the host. Once inside, the EB differentiates into the reticulate body (RB). The RB then undergoes multiple rounds of replication, before differentiating back to the infectious EB form. This cycle is essential for chlamydial survival as failure to switch between cell types prevents either host invasion or replication. Limitations in genetic techniques due to the obligate intracellular nature of Chlamydia have hampered identification of the molecular mechanisms involved in the cell-type development. We designed a novel dual promoter-reporter plasmid system that, in conjunction with live-cell microscopy, allows for the visualization of cell type switching in real time. To identify genes involved in the regulation of cell-type development, the live-cell promoter-reporter system was leveraged for the development of a forward genetic approach by combining chemical mutagenesis of the dual reporter strain, imaging and tracking of Chlamydia with altered developmental kinetics, followed by clonal isolation of mutants. This forward genetic workflow is a flexible tool that can be modified for directed interrogation into a wide range of genetic pathways.


Assuntos
Chlamydia trachomatis/genética , Chlamydia trachomatis/isolamento & purificação , Genômica/métodos , Mutação/genética , Chlamydia trachomatis/crescimento & desenvolvimento , Análise de Dados , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Humanos , Cinética , Mutagênese/genética , Fenótipo , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes
7.
Adv Pharmacol ; 88: 1-33, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32416864

RESUMO

Allosteric modulation of GPCRs, especially metabotropic glutamate (mGlu) receptors, has become an important strategy for drug discovery. Positive and negative allosteric modulators (PAM, NAM) are widely reported for the mGlu receptor family with leads mostly originating by high-throughput screening followed by iterative medicinal chemistry. The progression of the field from mutagenesis and homology modeling to elaborate structure-enabled drug discovery is described. We detail how computational methods have delivered new chemical matter and revealed the functional details of PAM and NAM activity. The breakthrough in mGlu receptor 7-transmembrane (7TM) crystal structures enabled recent combined modeling and experimental studies to confirm common binding sites, interactions and the origins of ligand effect on functional activity. Focusing on allosteric modulation of the mGlu2 and mGlu5 receptors, similarities are seen that still accommodate the known differences in binding sites and SAR. This work reveals the promise of a methodical computational approach built upon deep analysis of 7TM receptor simulations and interpretation of results in the context of our current understanding of receptor function. A crucial aspect was the close collaboration between modeling and experiment necessary to build and interrogate the hypotheses.


Assuntos
Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/metabolismo , Regulação Alostérica , Sítio Alostérico , Animais , Humanos , Ligantes , Modelos Moleculares , Mutagênese/genética
8.
Nat Biotechnol ; 38(7): 883-891, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32433547

RESUMO

Applications of adenine base editors (ABEs) have been constrained by the limited compatibility of the deoxyadenosine deaminase component with Cas homologs other than SpCas9. We evolved the deaminase component of ABE7.10 using phage-assisted non-continuous and continuous evolution (PANCE and PACE), which resulted in ABE8e. ABE8e contains eight additional mutations that increase activity (kapp) 590-fold compared with that of ABE7.10. ABE8e offers substantially improved editing efficiencies when paired with a variety of Cas9 or Cas12 homologs. ABE8e is more processive than ABE7.10, which could benefit screening, disruption of regulatory regions and multiplex base editing applications. A modest increase in Cas9-dependent and -independent DNA off-target editing, and in transcriptome-wide RNA off-target editing can be ameliorated by the introduction of an additional mutation in the TadA-8e domain. Finally, we show that ABE8e can efficiently install natural mutations that upregulate fetal hemoglobin expression in the BCL11A enhancer or in the the HBG promoter in human cells, targets that were poorly edited with ABE7.10. ABE8e augments the effectiveness and applicability of adenine base editing.


Assuntos
Adenina/metabolismo , Sistemas CRISPR-Cas/genética , DNA/genética , RNA/genética , Adenosina Desaminase/genética , Bacteriófagos/genética , Edição de Genes , Células HEK293 , Humanos , Mutagênese/genética , Mutação/genética
9.
Virus Res ; 283: 197976, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: covidwho-46070

RESUMO

An outbreak of atypical pneumonia caused by a novel Betacoronavirus (ßCoV), named SARS-CoV-2 has been declared a public health emergency of international concern by the World Health Organization. In order to gain insight into the emergence, evolution and adaptation of SARS-CoV-2 viruses, a comprehensive analysis of genome composition and codon usage of ßCoV circulating in China was performed. A biased nucleotide composition was found for SARS-CoV-2 genome. This bias in genomic composition is reflected in its codon and amino acid usage patterns. The overall codon usage in SARS-CoV-2 is similar among themselves and slightly biased. Most of the highly frequent codons are A- and U-ending, which strongly suggests that mutational bias is the main force shaping codon usage in this virus. Significant differences in relative synonymous codon usage frequencies among SARS-CoV-2 and human cells were found. These differences are due to codon usage preferences.


Assuntos
Betacoronavirus/classificação , Betacoronavirus/genética , Uso do Códon/genética , Doenças Transmissíveis Emergentes/virologia , Regulação Viral da Expressão Gênica/genética , Genoma Viral/genética , Genômica , Aminoácidos/genética , Animais , Betacoronavirus/isolamento & purificação , China/epidemiologia , Quirópteros/virologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Evolução Molecular , Furões/virologia , Humanos , Mutagênese/genética , Fases de Leitura Aberta/genética , Viverridae/virologia
10.
Nat Commun ; 11(1): 1848, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32296061

RESUMO

Genetically encoded Förster Resonance Energy Transfer (FRET)-based biosensors are powerful tools to illuminate spatiotemporal regulation of cell signaling in living cells, but the utility of the red spectrum for biosensing was limited due to a lack of bright and stable red fluorescent proteins. Here, we rationally improve the photophysical characteristics of the coral-derived fluorescent protein TagRFP-T. We show that a new single-residue mutant, super-TagRFP (stagRFP) has nearly twice the molecular brightness of TagRFP-T and negligible photoactivation. stagRFP facilitates significant improvements on multiple green-red biosensors as a FRET acceptor and is an efficient FRET donor that supports red/far-red FRET biosensing. Capitalizing on the ability of stagRFP to couple with multiple FRET partners, we develop a novel multiplex method to examine the confluence of signaling activities from three kinases simultaneously in single living cells, providing evidence for a role of Src family kinases in regulating growth factor induced Akt and ERK activities.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas Luminescentes/química , Humanos , Mutagênese/genética , Mutagênese/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
11.
Nat Commun ; 11(1): 2052, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345976

RESUMO

Cytosine base editors (CBEs) enable efficient, programmable reversion of T•A to C•G point mutations in the human genome. Recently, cytosine base editors with rAPOBEC1 were reported to induce unguided cytosine deamination in genomic DNA and cellular RNA. Here we report eight next-generation CBEs (BE4 with either RrA3F [wt, F130L], AmAPOBEC1, SsAPOBEC3B [wt, R54Q], or PpAPOBEC1 [wt, H122A, R33A]) that display comparable DNA on-target editing frequencies, whilst eliciting a 12- to 69-fold reduction in C-to-U edits in the transcriptome, and up to a 45-fold overall reduction in unguided off-target DNA deamination relative to BE4 containing rAPOBEC1. Further, no enrichment of genome-wide C•G to T•A edits are observed in mammalian cells following transfection of mRNA encoding five of these next-generation editors. Taken together, these next-generation CBEs represent a collection of base editing tools for applications in which minimized off-target and high on-target activity are required.


Assuntos
Citosina/metabolismo , DNA/genética , Edição de Genes , RNA/genética , Desaminase APOBEC-1/metabolismo , Citosina Desaminase/metabolismo , Replicação do DNA/genética , Desaminação , Genoma , Células HEK293 , Humanos , Mutagênese/genética , Transcrição Genética , Transcriptoma/genética
12.
J Vis Exp ; (157)2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32281972

RESUMO

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase variants that have more stringent specificity than the parental enzyme. The protocol requires the creation of a library of expression selection cassettes (ESCs) for variants of the REase, ideally with variability in positions likely to affect DNA binding. The ESC is flanked on one side by a sequence for the restriction site activity desired and a biotin tag and on the other side by a restriction site for the undesired activity and a primer annealing site. The ESCs are transcribed and translated in a water-in-oil emulsion, in conditions that make the presence of more than one DNA molecule per droplet unlikely. Therefore, the DNA in each cassette molecule is subjected only to the activity of the translated, encoded enzyme. REase variants of the desired specificity remove the biotin tag but not the primer annealing site. After breaking the emulsion, the DNA molecules are subjected to a biotin pulldown, and only those in the supernatant are retained. This step assures that only ESCs for variants that have not lost the desired activity are retained. These DNA molecules are then subjected to a first PCR reaction. Cleavage in the undesired sequence cuts off the primer binding site for one of the primers. Therefore, PCR amplifies only ESCs from droplets without the undesired activity. A second PCR reaction is then carried out to reintroduce the restriction site for the desired specificity and the biotin tag, so that the selection step can be reiterated. Selected open reading frames can be overexpressed in bacterial cells that also express the cognate methyltransferase of the parental REase, because the newly evolved REase targets only a subset of the methyltransferase target sites.


Assuntos
Enzimas de Restrição do DNA/metabolismo , Evolução Molecular Direcionada , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA/metabolismo , Enzimas de Restrição do DNA/química , Emulsões/química , Expressão Gênica , Mutagênese/genética , Óleos/química , Biossíntese de Proteínas , Engenharia de Proteínas , Especificidade por Substrato , Transcrição Genética , Água/química
13.
Proc Natl Acad Sci U S A ; 117(18): 9973-9980, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32303657

RESUMO

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeastlike fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 (rap/FK506) and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37° compared to 30° in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNA interference (RNAi) pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30° to the level observed at 37°. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host.


Assuntos
Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Micoses/genética , Retroelementos/genética , Animais , Antifúngicos/efeitos adversos , Cryptococcus neoformans/patogenicidade , Farmacorresistência Fúngica/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos , Mutagênese/genética , Micoses/microbiologia , Ácido Orótico/efeitos adversos , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Sirolimo/farmacologia , Tacrolimo/farmacologia , Virulência/genética
14.
Mutat Res ; 850-851: 503132, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32247550

RESUMO

EEMS and its successor Society EEMGS have provided a dynamic and successful platform to stimulate research and exchanges among the different actors involved in the protection of the environment and of human health from exposure to genome stressors. It includes basic, translational and applied research projects. This was possible due to the enthusiasm, creativity and support of scientists convinced of the importance of these issues. In the future young scientists will take over with new questions, new challenges, new technologies, new discoveries and new applications. A major challenge is the ethical questions emerging from the impressive potential of present genetic technologies capable of impacting the evolution of nature and humankind. The EEMGS, where academics, regulators and industries meet, should play a central role in these aspects, in particular in support of primary prevention and the establishment of internationally recognized guidelines. Collaboration with colleagues and other teams are of great importance to establish a stimulating open dialogue on scientific questions. However the key issues remain to do careful and rigorous research; to use logic and background knowledge; to define adequate experimental designs; to provide transparency in the protocols; to check repeatability of the results and to combine several statistical approaches in the quest to get to the truth. Among the many challenges ahead, re-evaluation of some key fundamental questions is necessary, such as the interplay between genetics and epigenetics, the existence of specific germ cell mutagens or the identification of the mechanisms leading to mutagen induced diseases. Translational and applied research will further include the development of systemic biomonitoring protocols, if possible in a single biological sample, the redaction of internationally harmonized guidelines but also the organization of platforms between geneticists and physicians open to all actors in the field. The creation of an independent European center to assess risk from exposure to mutagens, in particular in the light of the problematic of global warming might be very helpful.


Assuntos
Monitoramento Ambiental , Genoma Humano/genética , Metagenômica/tendências , Mutagênese/genética , Pesquisa Biomédica/tendências , Europa (Continente) , Humanos , Sociedades Científicas/tendências
15.
Mutat Res ; 850-851: 503136, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32247553

RESUMO

Tumorigenesis induced by oxidative stress is thought to be initiated by mutagenesis, but via an indirect mechanism. The dose-response curves for agents that act by this route usually show a threshold, for unknown reasons. To gain insight into these phenomena, we have analyzed the dose response for mutagenesis induced by the oral administration of potassium bromate, a typical oxidative-stress-generating agent, to gpt delta mice. The agent was given orally for 90 d to either Nrf2+ or Nrf2-knockout (KO) mice and mutants induced in the small intestine were analyzed. In Nrf2+mice, the mutant frequency was significantly greater than in the vehicle controls at a dose of 0.6 g/L but not at 0.2 g/L, indicating that a practical threshold for mutagenesis lies between these doses. At 0.6 g/L, the frequencies of G-to-T transversions (landmark mutations for oxidative stress) and G-to-A transitions were significantly elevated. In Nrf2-KO mice, too, the total mutant frequency was increased only at 0.6 g/L. G-to-T transversions are likely to have driven tumorigenesis in the small intestine. A site-specific G-to-T transversion at guanine (nucleotide 406) in a 5'-TGAA-3' sequence in gpt, and our primer extension reaction showed that formation of the oxidative DNA base modification 8-oxo-deoxyguanosine (8-oxo-dG) at nucleotide 406 was significantly increased at doses of 0.6 and 2 g/L in the gpt delta mice. In the Apc oncogene, guanine residues in the same or similar sequences (TGAA or AGAA) are highly substituted by thymine (G-to-T transversions) in potassium bromate-induced tumors. We propose that formation of 8-oxo-dG in the T(A)GAA sequence is an initiating event in tumor formation in the small intestine in response to oxidative stress.


Assuntos
Bromatos/toxicidade , Mutagênese/genética , Estresse Oxidativo/genética , Pentosiltransferases/genética , 8-Hidroxi-2'-Desoxiguanosina/genética , Administração Oral , Animais , Bromatos/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , DNA/efeitos dos fármacos , DNA/genética , Relação Dose-Resposta a Droga , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/patologia , Camundongos , Camundongos Knockout , Mutagênese/efeitos dos fármacos , Mutação , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos
16.
Virus Res ; 283: 197976, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32294518

RESUMO

An outbreak of atypical pneumonia caused by a novel Betacoronavirus (ßCoV), named SARS-CoV-2 has been declared a public health emergency of international concern by the World Health Organization. In order to gain insight into the emergence, evolution and adaptation of SARS-CoV-2 viruses, a comprehensive analysis of genome composition and codon usage of ßCoV circulating in China was performed. A biased nucleotide composition was found for SARS-CoV-2 genome. This bias in genomic composition is reflected in its codon and amino acid usage patterns. The overall codon usage in SARS-CoV-2 is similar among themselves and slightly biased. Most of the highly frequent codons are A- and U-ending, which strongly suggests that mutational bias is the main force shaping codon usage in this virus. Significant differences in relative synonymous codon usage frequencies among SARS-CoV-2 and human cells were found. These differences are due to codon usage preferences.


Assuntos
Betacoronavirus/classificação , Betacoronavirus/genética , Uso do Códon/genética , Doenças Transmissíveis Emergentes/virologia , Regulação Viral da Expressão Gênica/genética , Genoma Viral/genética , Genômica , Aminoácidos/genética , Animais , Betacoronavirus/isolamento & purificação , China/epidemiologia , Quirópteros/virologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Evolução Molecular , Furões/virologia , Humanos , Mutagênese/genética , Fases de Leitura Aberta/genética , Viverridae/virologia
17.
Infect Immun ; 88(5)2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32152196

RESUMO

The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia's ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.


Assuntos
Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/genética , Chlamydia trachomatis/patogenicidade , Mutagênese/genética , Actinas/genética , Alelos , Animais , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Fluorescência , Deleção de Genes , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C3H , Fosfoproteínas/genética , Virulência/genética
19.
Nature ; 578(7793): 82-93, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32025007

RESUMO

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale1-3. Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter4; identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation5,6; analyses timings and patterns of tumour evolution7; describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity8,9; and evaluates a range of more-specialized features of cancer genomes8,10-18.


Assuntos
Análise Mutacional de DNA , Evolução Molecular , Genoma Humano/genética , Genômica , Mutação , Neoplasias/genética , Proliferação de Células/genética , Senescência Celular/genética , Cromotripsia , Computação em Nuvem , Feminino , Mutação em Linhagem Germinativa/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Disseminação de Informação , Masculino , Mutagênese/genética , Neoplasias/classificação , Neoplasias/patologia , Oncogenes/genética , Regiões Promotoras Genéticas/genética , Processamento de RNA/genética , Reprodutibilidade dos Testes , Telomerase/genética , Telômero/genética
20.
J Vis Exp ; (155)2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-32065121

RESUMO

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as axolotls, to regenerate entire limbs as adults. Here, we outline techniques used to generate chimeric axolotls with Cas9-mutagenized haploid forelimbs that can be used for exploring gene function and the fidelity of limb regeneration. We combine several embryological and genetic techniques, including haploid generation via in vitro activation, CRISPR/Cas9 mutagenesis, and tissue grafting into one protocol to produce a unique system for haploid genetic screening in a model organism of regeneration. This strategy reduces the number of animals, space, and time required for the functional analysis of genes in limb regeneration. This also permits the investigation of regeneration-specific functions of genes that may be required for other essential processes, such as organogenesis, tissue morphogenesis, and other essential embryonic processes. The method described here is a unique platform for conducting haploid genetic screening in a vertebrate model system.


Assuntos
Ambystoma mexicanum/embriologia , Ambystoma mexicanum/genética , Quimera/genética , Extremidades/embriologia , Haploidia , Mutação/genética , Animais , Diploide , Embrião não Mamífero/metabolismo , Feminino , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Masculino , Mutagênese/genética , Fenótipo , Regeneração/genética
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