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1.
Nat Commun ; 11(1): 4886, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985500

RESUMO

Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations, including the two most frequent 52 bp deletion (del52) and 5 bp insertion (ins5), induce a frameshift to the same alternative reading frame generating new C-terminal tails. In patients, del52 and ins5 induce two phenotypically distinct myeloproliferative neoplasms (MPNs). They are equally found in ET, but del52 is more frequent in PMF. We generated heterozygous and homozygous conditional inducible knock-in (KI) mice expressing a chimeric murine CALR del52 or ins5 with the human mutated C-terminal tail to investigate their pathogenic effects on hematopoiesis. Del52 induces greater phenotypic changes than ins5 including thrombocytosis, leukocytosis, splenomegaly, bone marrow hypocellularity, megakaryocytic lineage amplification, expansion and competitive advantage of the hematopoietic stem cell compartment. Homozygosity amplifies these features, suggesting a distinct contribution of homozygous clones to human MPNs. Moreover, homozygous del52 KI mice display features of a penetrant myelofibrosis-like disorder with extramedullary hematopoiesis linked to splenomegaly, megakaryocyte hyperplasia and the presence of reticulin fibers. Overall, modeling del52 and ins5 mutations in mice successfully recapitulates the differences in phenotypes observed in patients.


Assuntos
Calreticulina/genética , Mielofibrose Primária/genética , Trombocitemia Essencial/genética , Animais , Calreticulina/metabolismo , Modelos Animais de Doenças , Feminino , Células-Tronco Hematopoéticas/metabolismo , Homozigoto , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Fenótipo , Mielofibrose Primária/metabolismo , Deleção de Sequência , Trombocitemia Essencial/metabolismo
2.
Nat Protoc ; 15(8): 2705-2727, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32681154

RESUMO

Invasive fungal infections caused by Candida species are life threatening with high mortality, posing a severe public health threat. New technologies for rapid, genome-wide identification of virulence genes and therapeutic targets are urgently needed. Our recent engineering of a piggyBac (PB) transposon-mediated mutagenesis system in haploid Candida albicans provides a powerful discovery tool, which we anticipate should be adaptable to other haploid Candida species. In this protocol, we use haploid C. albicans as an example to present an improved version of the mutagenesis system and provide a detailed description of the protocol for constructing high-quality mutant libraries. We also describe a method for quantitative PB insertion site sequencing, PBISeq. The PBISeq library preparation procedure exploits tagmentation to quickly and efficiently construct sequencing libraries. Finally, we present a pipeline to analyze PB insertion sites in a de novo assembled genome of our engineered haploid C. albicans strain. The entire protocol takes ~7 d from transposition induction to having a final library ready for sequencing. This protocol is highly efficient and less labor intensive than alternative approaches and significantly accelerates genetic studies of Candida.


Assuntos
Candida/genética , Elementos de DNA Transponíveis/genética , Genoma Fúngico/genética , Haploidia , Mutagênese Insercional/métodos
3.
Proc Natl Acad Sci U S A ; 117(32): 19328-19338, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32690705

RESUMO

Co-option of transposable elements (TEs) to become part of existing or new enhancers is an important mechanism for evolution of gene regulation. However, contributions of lineage-specific TE insertions to recent regulatory adaptations remain poorly understood. Gibbons present a suitable model to study these contributions as they have evolved a lineage-specific TE called LAVA (LINE-AluSz-VNTR-Alu LIKE), which is still active in the gibbon genome. The LAVA retrotransposon is thought to have played a role in the emergence of the highly rearranged structure of the gibbon genome by disrupting transcription of cell cycle genes. In this study, we investigated whether LAVA may have also contributed to the evolution of gene regulation by adopting enhancer function. We characterized fixed and polymorphic LAVA insertions across multiple gibbons and found 96 LAVA elements overlapping enhancer chromatin states. Moreover, LAVA was enriched in multiple transcription factor binding motifs, was bound by an important transcription factor (PU.1), and was associated with higher levels of gene expression in cis We found gibbon-specific signatures of purifying/positive selection at 27 LAVA insertions. Two of these insertions were fixed in the gibbon lineage and overlapped with enhancer chromatin states, representing putative co-opted LAVA enhancers. These putative enhancers were located within genes encoding SETD2 and RAD9A, two proteins that facilitate accurate repair of DNA double-strand breaks and prevent chromosomal rearrangement mutations. Co-option of LAVA in these genes may have influenced regulation of processes that preserve genome integrity. Our findings highlight the importance of considering lineage-specific TEs in studying evolution of gene regulatory elements.


Assuntos
Genoma , Hylobates/genética , Retroelementos , Animais , Cromatina/genética , Evolução Molecular , Regulação da Expressão Gênica , Hylobates/classificação , Mutagênese Insercional , Sequências Reguladoras de Ácido Nucleico , Especificidade da Espécie
4.
PLoS Comput Biol ; 16(7): e1007980, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32678849

RESUMO

Bacteria need to survive in a wide range of environments. Currently, there is an incomplete understanding of the genetic basis for mechanisms underpinning survival in stressful conditions, such as the presence of anti-microbials. Transposon directed insertion-site sequencing (TraDIS) is a powerful tool to identify genes and networks which are involved in survival and fitness under a given condition by simultaneously assaying the fitness of millions of mutants, thereby relating genotype to phenotype and contributing to an understanding of bacterial cell biology. A recent refinement of this approach allows the roles of essential genes in conditional stress survival to be inferred by altering their expression. These advancements combined with the rapidly falling costs of sequencing now allows comparisons between multiple experiments to identify commonalities in stress responses to different conditions. This capacity however poses a new challenge for analysis of multiple data sets in conjunction. To address this analysis need, we have developed 'AlbaTraDIS'; a software application for rapid large-scale comparative analysis of TraDIS experiments that predicts the impact of transposon insertions on nearby genes. AlbaTraDIS can identify genes which are up or down regulated, or inactivated, between multiple conditions, producing a filtered list of genes for further experimental validation as well as several accompanying data visualisations. We demonstrate the utility of our new approach by applying it to identify genes used by Escherichia coli to survive in a wide range of different concentrations of the biocide Triclosan. AlbaTraDIS identified all well characterised Triclosan resistance genes, including the primary target, fabI. A number of new loci were also implicated in Triclosan resistance and the predicted phenotypes for a selection of these were validated experimentally with results being consistent with predictions. AlbaTraDIS provides a simple and rapid method to analyse multiple transposon mutagenesis data sets allowing this technology to be used at large scale. To our knowledge this is the only tool currently available that can perform these tasks. AlbaTraDIS is written in Python 3 and is available under the open source licence GNU GPL 3 from https://github.com/quadram-institute-bioscience/albatradis.


Assuntos
Biologia Computacional , Elementos de DNA Transponíveis , Escherichia coli/genética , Mutagênese Insercional , Software , Algoritmos , Anti-Infecciosos/farmacologia , Farmacorresistência Bacteriana , Biblioteca Gênica , Genes Essenciais , Genoma Bacteriano , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Fenótipo , Biossíntese de Proteínas , Triclosan/farmacologia
5.
Proc Natl Acad Sci U S A ; 117(26): 15193-15199, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32522874

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an immediate, major threat to public health across the globe. Here we report an in-depth molecular analysis to reconstruct the evolutionary origins of the enhanced pathogenicity of SARS-CoV-2 and other coronaviruses that are severe human pathogens. Using integrated comparative genomics and machine learning techniques, we identify key genomic features that differentiate SARS-CoV-2 and the viruses behind the two previous deadly coronavirus outbreaks, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), from less pathogenic coronaviruses. These features include enhancement of the nuclear localization signals in the nucleocapsid protein and distinct inserts in the spike glycoprotein that appear to be associated with high case fatality rate of these coronaviruses as well as the host switch from animals to humans. The identified features could be crucial contributors to coronavirus pathogenicity and possible targets for diagnostics, prognostication, and interventions.


Assuntos
Betacoronavirus/genética , Evolução Molecular , Genoma Viral , Proteínas do Nucleocapsídeo/genética , Glicoproteína da Espícula de Coronavírus/genética , Animais , Betacoronavirus/classificação , Betacoronavirus/patogenicidade , Especificidade de Hospedeiro , Humanos , Aprendizado de Máquina , Coronavírus da Síndrome Respiratória do Oriente Médio/classificação , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Mutagênese Insercional , Sinais de Localização Nuclear/genética , Proteínas do Nucleocapsídeo/química , Filogenia , Homologia de Sequência , Glicoproteína da Espícula de Coronavírus/química , Virulência/genética
6.
Curr Biol ; 30(11): 2196-2203.e3, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: covidwho-232567

RESUMO

The unprecedented pandemic of pneumonia caused by a novel coronavirus, SARS-CoV-2, in China and beyond has had major public health impacts on a global scale [1, 2]. Although bats are regarded as the most likely natural hosts for SARS-CoV-2 [3], the origins of the virus remain unclear. Here, we report a novel bat-derived coronavirus, denoted RmYN02, identified from a metagenomic analysis of samples from 227 bats collected from Yunnan Province in China between May and October 2019. Notably, RmYN02 shares 93.3% nucleotide identity with SARS-CoV-2 at the scale of the complete virus genome and 97.2% identity in the 1ab gene, in which it is the closest relative of SARS-CoV-2 reported to date. In contrast, RmYN02 showed low sequence identity (61.3%) to SARS-CoV-2 in the receptor-binding domain (RBD) and might not bind to angiotensin-converting enzyme 2 (ACE2). Critically, and in a similar manner to SARS-CoV-2, RmYN02 was characterized by the insertion of multiple amino acids at the junction site of the S1 and S2 subunits of the spike (S) protein. This provides strong evidence that such insertion events can occur naturally in animal betacoronaviruses.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Quirópteros/virologia , Mutagênese Insercional , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Animais , Betacoronavirus/química , Eutérios/virologia , Fezes/virologia , Genoma Viral , Modelos Moleculares , Filogenia , RNA Viral/genética , Vírus da SARS/genética , Alinhamento de Sequência , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/química
7.
PLoS One ; 15(5): e0232594, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32401787

RESUMO

Generation and subsequent analysis of mutants is critical to understanding the functions of genes and proteins. Here we describe TIM, an efficient, cost-effective, CRISPR-based targeted insertional mutagenesis method for the model organism Chlamydomonas reinhardtii. TIM utilizes delivery into the cell of a Cas9-guide RNA (gRNA) ribonucleoprotein (RNP) together with exogenous double-stranded (donor) DNA. The donor DNA contains gene-specific homology arms and an integral antibiotic-resistance gene that inserts at the double-stranded break generated by Cas9. After optimizing multiple parameters of this method, we were able to generate mutants for six out of six different genes in two different cell-walled strains with mutation efficiencies ranging from 40% to 95%. Furthermore, these high efficiencies allowed simultaneous targeting of two separate genes in a single experiment. TIM is flexible with regard to many parameters and can be carried out using either electroporation or the glass-bead method for delivery of the RNP and donor DNA. TIM achieves a far higher mutation rate than any previously reported for CRISPR-based methods in C. reinhardtii and promises to be effective for many, if not all, non-essential nuclear genes.


Assuntos
Sistemas CRISPR-Cas , Chlamydomonas reinhardtii/genética , Edição de Genes/métodos , Mutagênese Insercional/métodos , DNA/genética , RNA Guia/genética
8.
Curr Biol ; 30(11): 2196-2203.e3, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32416074

RESUMO

The unprecedented pandemic of pneumonia caused by a novel coronavirus, SARS-CoV-2, in China and beyond has had major public health impacts on a global scale [1, 2]. Although bats are regarded as the most likely natural hosts for SARS-CoV-2 [3], the origins of the virus remain unclear. Here, we report a novel bat-derived coronavirus, denoted RmYN02, identified from a metagenomic analysis of samples from 227 bats collected from Yunnan Province in China between May and October 2019. Notably, RmYN02 shares 93.3% nucleotide identity with SARS-CoV-2 at the scale of the complete virus genome and 97.2% identity in the 1ab gene, in which it is the closest relative of SARS-CoV-2 reported to date. In contrast, RmYN02 showed low sequence identity (61.3%) to SARS-CoV-2 in the receptor-binding domain (RBD) and might not bind to angiotensin-converting enzyme 2 (ACE2). Critically, and in a similar manner to SARS-CoV-2, RmYN02 was characterized by the insertion of multiple amino acids at the junction site of the S1 and S2 subunits of the spike (S) protein. This provides strong evidence that such insertion events can occur naturally in animal betacoronaviruses.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Quirópteros/virologia , Mutagênese Insercional , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Animais , Betacoronavirus/química , Eutérios/virologia , Fezes/virologia , Genoma Viral , Modelos Moleculares , Filogenia , RNA Viral/genética , Vírus da SARS/genética , Alinhamento de Sequência , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/química
9.
Nat Chem Biol ; 16(7): 725-730, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32284602

RESUMO

Anti-CRISPR (Acr) proteins are powerful tools to control CRISPR-Cas technologies. However, the available Acr repertoire is limited to naturally occurring variants. Here, we applied structure-based design on AcrIIC1, a broad-spectrum CRISPR-Cas9 inhibitor, to improve its efficacy on different targets. We first show that inserting exogenous protein domains into a selected AcrIIC1 surface site dramatically enhances inhibition of Neisseria meningitidis (Nme)Cas9. Then, applying structure-guided design to the Cas9-binding surface, we converted AcrIIC1 into AcrIIC1X, a potent inhibitor of the Staphylococcus aureus (Sau)Cas9, an orthologue widely applied for in vivo genome editing. Finally, to demonstrate the utility of AcrIIC1X for genome engineering applications, we implemented a hepatocyte-specific SauCas9 ON-switch by placing AcrIIC1X expression under regulation of microRNA-122. Our work introduces designer Acrs as important biotechnological tools and provides an innovative strategy to safeguard CRISPR technologies.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , MicroRNAs/genética , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Genoma Humano , Células HEK293 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , MicroRNAs/metabolismo , Modelos Moleculares , Mutagênese Insercional , Neisseria meningitidis/enzimologia , Neisseria meningitidis/genética , Plasmídeos/química , Plasmídeos/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , RNA Guia/genética , RNA Guia/metabolismo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética
10.
PLoS One ; 15(4): e0231323, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32287287

RESUMO

Following allopolyploidization, nascent polyploid wheat species react with massive genomic rearrangements, including deletion of transposable element-containing sequences. While such massive rearrangements are considered to be a prominent process in wheat genome evolution and speciation, their structure, extent, and underlying mechanisms remain poorly understood. In this study, we retrieved ~3500 insertions of a specific variant of Fatima, one of the most dynamic gypsy long-terminal repeat retrotransposons in wheat from the recently available high-quality genome drafts of Triticum aestivum (bread wheat) and Triticum turgidum ssp. dicoccoides or wild emmer, the allotetraploid mother of all modern wheats. The dynamic nature of Fatima facilitated the identification of large (i.e., up to ~ 1 million bases) Fatima-containing insertions/deletions (indels) upon comparison of bread wheat and wild emmer genomes. We characterized 11 such indels using computer-assisted analysis followed by PCR validation, and found that they might have occurred via unequal intra-strand recombination or double-strand break (DSB) events. Additionally, we observed one case of introgression of novel DNA fragments from an unknown source into the wheat genome. Our data thus indicate that massive large-scale DNA rearrangements might play a prominent role in wheat speciation.


Assuntos
Evolução Molecular , Rearranjo Gênico , Genoma de Planta , Triticum/genética , Quebras de DNA de Cadeia Dupla , Variações do Número de Cópias de DNA , Deleção de Genes , Mutagênese Insercional , Recombinação Genética , Retroelementos/genética
11.
Nat Commun ; 11(1): 1796, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286261

RESUMO

Chromatin looping is important for gene regulation, and studies of 3D chromatin structure across species and cell types have improved our understanding of the principles governing chromatin looping. However, 3D genome evolution and its relationship with natural selection remains largely unexplored. In mammals, the CTCF protein defines the boundaries of most chromatin loops, and variations in CTCF occupancy are associated with looping divergence. While many CTCF binding sites fall within transposable elements (TEs), their contribution to 3D chromatin structural evolution is unknown. Here we report the relative contributions of TE-driven CTCF binding site expansions to conserved and divergent chromatin looping in human and mouse. We demonstrate that TE-derived CTCF binding divergence may explain a large fraction of variable loops. These variable loops contribute significantly to corresponding gene expression variability across cells and species, possibly by refining sub-TAD-scale loop contacts responsible for cell-type-specific enhancer-promoter interactions.


Assuntos
Cromatina/metabolismo , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica , Genoma , Mamíferos/genética , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromossomos de Mamíferos/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Mutagênese Insercional/genética , Conformação de Ácido Nucleico , Filogenia , Especificidade da Espécie , Sintenia/genética
12.
PLoS Genet ; 16(4): e1008462, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32236090

RESUMO

In flowering plants, gene expression in the haploid male gametophyte (pollen) is essential for sperm delivery and double fertilization. Pollen also undergoes dynamic epigenetic regulation of expression from transposable elements (TEs), but how this process interacts with gene expression is not clearly understood. To explore relationships among these processes, we quantified transcript levels in four male reproductive stages of maize (tassel primordia, microspores, mature pollen, and sperm cells) via RNA-seq. We found that, in contrast with vegetative cell-limited TE expression in Arabidopsis pollen, TE transcripts in maize accumulate as early as the microspore stage and are also present in sperm cells. Intriguingly, coordinate expression was observed between highly expressed protein-coding genes and their neighboring TEs, specifically in mature pollen and sperm cells. To investigate a potential relationship between elevated gene transcript level and pollen function, we measured the fitness cost (male-specific transmission defect) of GFP-tagged coding sequence insertion mutations in over 50 genes identified as highly expressed in the pollen vegetative cell, sperm cell, or seedling (as a sporophytic control). Insertions in seedling genes or sperm cell genes (with one exception) exhibited no difference from the expected 1:1 transmission ratio. In contrast, insertions in over 20% of vegetative cell genes were associated with significant reductions in fitness, showing a positive correlation of transcript level with non-Mendelian segregation when mutant. Insertions in maize gamete expressed2 (Zm gex2), the sole sperm cell gene with measured contributions to fitness, also triggered seed defects when crossed as a male, indicating a conserved role in double fertilization, given the similar phenotype previously demonstrated for the Arabidopsis ortholog GEX2. Overall, our study demonstrates a developmentally programmed and coordinated transcriptional activation of TEs and genes in pollen, and further identifies maize pollen as a model in which transcriptomic data have predictive value for quantitative phenotypes.


Assuntos
Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica de Plantas , Aptidão Genética , Pólen/genética , Transcrição Genética , Zea mays/genética , Linhagem da Célula , Perfilação da Expressão Gênica , Genes de Plantas/genética , Genoma de Planta/genética , Meiose , Mutagênese Insercional , Mutação , Polinização , Reprodutibilidade dos Testes , Reprodução , Sementes/genética , Sementes/crescimento & desenvolvimento , Regulação para Cima , Zea mays/citologia , Zea mays/crescimento & desenvolvimento
13.
F1000Res ; 92020.
Artigo em Inglês | MEDLINE | ID: mdl-32148769

RESUMO

Since Barbara McClintock's groundbreaking discovery of mobile DNA sequences some 70 years ago, transposable elements have come to be recognized as important mutagenic agents impacting genome composition, genome evolution, and human health. Transposable elements are a major constituent of prokaryotic and eukaryotic genomes, and the transposition mechanisms enabling transposon proliferation over evolutionary time remain engaging topics for study, suggesting complex interactions with the host, both antagonistic and mutualistic. The impact of transposition is profound, as over 100 human heritable diseases have been attributed to transposon insertions. Transposition can be highly mutagenic, perturbing genome integrity and gene expression in a wide range of organisms. This mutagenic potential has been exploited in the laboratory, where transposons have long been utilized for phenotypic screening and the generation of defined mutant libraries. More recently, barcoding applications and methods for RNA-directed transposition are being used towards new phenotypic screens and studies relevant for gene therapy. Thus, transposable elements are significant in affecting biology both in vivo and in the laboratory, and this review will survey advances in understanding the biological role of transposons and relevant laboratory applications of these powerful molecular tools.


Assuntos
Elementos de DNA Transponíveis , Mutagênese Insercional , Código de Barras de DNA Taxonômico , Terapia Genética , Humanos
14.
Biochim Biophys Acta Proteins Proteom ; 1868(7): 140411, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32200007

RESUMO

Calpain-3 (CAPN3), a 94-kDa member of the calpain protease family, is abundant in skeletal muscle. Mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A, indicating that CAPN3 plays important roles in muscle physiology. CAPN3 has several unique features. A crystallographic study revealed that its C-terminal penta-EF-hand domains form a homodimer, suggesting that CAPN3 functions as a homodimeric protease. To analyze complex formation of CAPN3 in a more convenient manner, we performed blue native polyacrylamide gel electrophoresis and found that the observed molecular weight of native CAPN3, as well as recombinant CAPN3, was larger than 240 kDa. Further analysis by cross-linking and sequential immunoprecipitation revealed that CAPN3 in fact forms a homotrimer. Trimer formation was abolished by the deletion of the PEF domain, but not the CAPN3-specific insertion sequences NS, IS1, and IS2. The PEF domain alone formed a homodimer, as reported, but addition of the adjacent CBSW domain to its N-terminus reinforced the trimer-forming property. Collectively, these results suggest that CAPN3 forms a homotrimer in which the PEF domain's dimer-forming ability is influenced by other domains.


Assuntos
Calpaína/metabolismo , Proteínas Musculares/metabolismo , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Animais , Calpaína/química , Calpaína/genética , Linhagem Celular , Motivos EF Hand , Feminino , Predisposição Genética para Doença/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/química , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Mutagênese Insercional , Mutação , Domínios Proteicos
15.
J Immunol ; 204(7): 1982-1987, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32122998

RESUMO

GFP is frequently used as a marker for tracking donor cells adoptively transplanted into recipient animals. The human ubiquitin C promoter (UBC)-driven-GFP transgenic mouse is a commonly used source of donor cells for this purpose. This mouse was initially generated in the C57BL/6 inbred strain and has been backcrossed into the BALB/cBy strain for over 11 generations. Both the C57BL/6 inbred and BALB/cBy congenic UBC-GFP lines are commercially available and have been widely distributed. These UBC-GFP lines can be a convenient resource for tracking donor cells in both syngenic MHC-matched and in allogenic MHC-mismatched studies as C57BL/6 (H-2b) and BALB/cBy (H-2d) have disparate MHC haplotypes. In this report, we surprisingly discover that the UBC-GFP BALB/cBy congenic mice still retain the H-2b MHC haplotype of their original C57BL/6 founder, suggesting that the UBC-GFP transgene integration site is closely linked to the MHC locus on chromosome 17. Using linear amplification-mediated PCR, we successfully map the UBC-GFP transgene to the MHC locus. This study highlights the importance and urgency of mapping the transgene integration site of transgenic mouse strains used in biomedical research. Furthermore, this study raises the possibility of alternative interpretations of previous studies using congenic UBC-GFP mice and focuses attention on the necessity for rigor and reproducibility in scientific research.


Assuntos
Cromossomos/genética , Proteínas de Fluorescência Verde/genética , Complexo Principal de Histocompatibilidade/genética , Mutagênese Insercional/genética , Transgenes/genética , Ubiquitina C/genética , Animais , Haplótipos/genética , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes
16.
PLoS One ; 15(2): e0229283, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084202

RESUMO

Necrotizing enterocolitis (NEC) is a devastating intestinal emergency that affects ten percent of very low birth weight premature babies and costs society in both expense and heartache. It is probably caused by an inappropriate interaction of colonizing bacteria with an immature intestine. A possible preventative measure is to feed prematures their mother's expressed breast milk in conjunction with a probiotic. This synbiotic prevention reduces the severity and incidence of this condition. This study was designed to determine the mechanism of the synbiotic effect in human and mouse fetal intestine. Breast milk interacting with a NEC preventative probiotic such as Bifidobacterium infantis can produce increased levels of short chain fatty acids (acetate, propionate and butyrate) (SCFAs). SCFAs are known to be anti-inflammatory in mature enterocytes and immunocytes. Very little is known about their role in immature intestine. When exposed to a human fetal cell line, fetal intestinal organoids and fetal mouse intestine, these SCFAs were anti-inflammatory. Their mechanism of anti-inflammation differed from those reported for mature cells by involving the G-protein coupled receptor (GPR 109A) and inhibiting histone deacetylase 4 and 5. These bacterial metabolites may help explain the synbiotic anti-inflammatory effect of breast milk and probiotics given to premature infants at risk for NEC.


Assuntos
Bifidobacterium longum subspecies infantis/fisiologia , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Ácidos Graxos Voláteis/biossíntese , Ácidos Graxos Voláteis/farmacologia , Intestinos/microbiologia , Leite Humano/microbiologia , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Enterócitos/metabolismo , Indução Enzimática/efeitos dos fármacos , Feto/microbiologia , Histona Desacetilases/genética , Humanos , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Intestinos/citologia , Camundongos , Mutagênese Insercional/efeitos dos fármacos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo
17.
Plant Cell Rep ; 39(4): 543-552, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32025802

RESUMO

KEY MESSAGE: BIG regulates the shoot stem cell population. The shoot apical meristem (SAM) contains a population of self-renewing cells, and provides daughter cells for initiation and development of aerial parts of plants. However, the underlying mechanisms of SAM size regulation remain largely unclear. Here, we identified a mutant that displayed a large SAM, designated big-shoot meristem (big-m), in Arabidopsis thaliana. The phenotype of big-m is caused by a new T-DNA insertion allele of BIG, causing a loss of function. The big-m mutant had more stem cells in the SAM than in the wild type. Expression of WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM) was promoted in big-m compared with the wild type, showing that BIG functions upstream of WUS and STM. Therefore, BIG is an important regulator of the stem cell population in the SAM. Furthermore, genetic analysis indicated that BIG acts synergistically with PIN-FORMED1 (PIN1) in controlling SAM size. Our results suggest that BIG plays an important role in controlling Arabidopsis thaliana SAM growth via PIN1-mediated auxin homeostasis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Meristema/citologia , Meristema/genética , Brotos de Planta/citologia , Brotos de Planta/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação a Calmodulina/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Mutagênese Insercional , Fenótipo , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo
19.
J Vis Exp ; (155)2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32065159

RESUMO

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.


Assuntos
Alelos , Chlamydia trachomatis/genética , Deleção de Genes , Mutagênese Insercional/genética , Mutagênese/genética , Sequência de Bases , DNA Bacteriano/genética , Genoma Bacteriano , Proteínas de Fluorescência Verde/genética , Integrases/metabolismo , Transformação Genética
20.
Nature ; 578(7793): 112-121, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32025012

RESUMO

A key mutational process in cancer is structural variation, in which rearrangements delete, amplify or reorder genomic segments that range in size from kilobases to whole chromosomes1-7. Here we develop methods to group, classify and describe somatic structural variants, using data from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), which aggregated whole-genome sequencing data from 2,658 cancers across 38 tumour types8. Sixteen signatures of structural variation emerged. Deletions have a multimodal size distribution, assort unevenly across tumour types and patients, are enriched in late-replicating regions and correlate with inversions. Tandem duplications also have a multimodal size distribution, but are enriched in early-replicating regions-as are unbalanced translocations. Replication-based mechanisms of rearrangement generate varied chromosomal structures with low-level copy-number gains and frequent inverted rearrangements. One prominent structure consists of 2-7 templates copied from distinct regions of the genome strung together within one locus. Such cycles of templated insertions correlate with tandem duplications, and-in liver cancer-frequently activate the telomerase gene TERT. A wide variety of rearrangement processes are active in cancer, which generate complex configurations of the genome upon which selection can act.


Assuntos
Variação Genética , Genoma Humano/genética , Neoplasias/genética , Rearranjo Gênico/genética , Genômica , Humanos , Mutagênese Insercional , Telomerase/genética
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