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1.
Nat Commun ; 12(1): 780, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594041

RESUMO

Novel pathogenic coronaviruses - such as SARS-CoV and probably SARS-CoV-2 - arise by homologous recombination between co-infecting viruses in a single cell. Identifying possible sources of novel coronaviruses therefore requires identifying hosts of multiple coronaviruses; however, most coronavirus-host interactions remain unknown. Here, by deploying a meta-ensemble of similarity learners from three complementary perspectives (viral, mammalian and network), we predict which mammals are hosts of multiple coronaviruses. We predict that there are 11.5-fold more coronavirus-host associations, over 30-fold more potential SARS-CoV-2 recombination hosts, and over 40-fold more host species with four or more different subgenera of coronaviruses than have been observed to date at >0.5 mean probability cut-off (2.4-, 4.25- and 9-fold, respectively, at >0.9821). Our results demonstrate the large underappreciation of the potential scale of novel coronavirus generation in wild and domesticated animals. We identify high-risk species for coronavirus surveillance.


Assuntos
Coronavirus/fisiologia , Interações Hospedeiro-Patógeno , Mamíferos/virologia , Animais , Infecções por Coronavirus/virologia , Humanos , Modelos Biológicos , Filogenia , Recombinação Genética/genética , Reprodutibilidade dos Testes
2.
Arch Virol ; 166(2): 491-499, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33394171

RESUMO

The family Tospoviridae of the order Bunyavirales is constituted of tri-segmented negative-sense single-stranded RNA viruses that infect plants and are also able to replicate in their insect vectors in a persistent manner. The family is composed of a single genus, Orthotospovirus, whose type species is Tomato spotted wilt orthotospovirus. Previous studies assessing the phylogenetic relationships within this genus were based on partial genomic sequences, resulting in unresolved clades and a poor assessment of the roles of recombination and segment reassortment during mixed infections. Full genome sequences of members of recognized Orthotospovirus species are now available at NCBI. In this study, we examined 67 complete genome sequences from members of 22 species. Our study confirms the existence of four phylogroups (A to D), grouped in two major clades (A-B and C-D) within the genus. We found strong evidence that within-segment recombination events and reassortment of segments during mixed infections have been involved in the origin of new orthotospoviruses. Also, selection pressures were analyzed for each gene, and evidence of positive selection was found in all genes.


Assuntos
Genoma Viral/genética , Recombinação Genética/genética , Tospovirus/genética , Infecções por Bunyaviridae/virologia , Lycopersicon esculentum/virologia , Filogenia , Doenças das Plantas/virologia , Sequenciamento Completo do Genoma/métodos
3.
Arch Virol ; 166(2): 389-402, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33385245

RESUMO

Recombination is an important phenomenon that accelerates evolution and enriches the genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV). Recombinant PRRSV isolates sometimes have different genetic backgrounds. In this study, we report a recombinant PRRSV (SD-YL1712) isolated from a pig farm. The genome of SD-YL1712 is 15,014 nucleotides in length, and its nucleotide and amino acid sequence conservation is higher than that of PRRSV strain JXA1 except within the NSP2 region. The NSP2 region of SDYL1712 shares the highest nucleotide (85.9%) and amino acid (84.1%) sequence identity with PRRSV strain NADC30. SD-YL1712 was found to contain a characteristic 131-amino-acid deletion in the NSP2 region. Two recombination breakpoints were detected at nt 2134 and nt 3958 within the NSP2 region, which revealed that SD-YL1712 originated from a recombination event between NADC30-like and HP-PRRSV-derived MLV-like strains. Interestingly, SD-YL1712 had an additional deletion at position 586, similar to that found in strain TJnh1501. Moreover, the pathogenicity of strain SD-YL1712 was found to be similar to that of HP-PRRSV JXA1, which was higher than that of the CH1a strain. Further analysis indicated that SD-YL1712 might be a transitional intermediate in the evolution of TJbd1401 to TJnh1501.


Assuntos
Genoma Viral/genética , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Recombinação Genética/genética , Sequência de Aminoácidos , Animais , China , Evolução Molecular , Fazendas , Variação Genética/genética , Genômica , Filogenia , Análise de Sequência de DNA/métodos , Suínos , Proteínas não Estruturais Virais/genética , Virulência/genética
4.
PLoS Genet ; 16(12): e1009272, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33332358

RESUMO

The Betacoronaviruses comprise multiple subgenera whose members have been implicated in human disease. As with SARS, MERS and now SARS-CoV-2, the origin and emergence of new variants are often attributed to events of recombination that alter host tropism or disease severity. In most cases, recombination has been detected by searches for excessively similar genomic regions in divergent strains; however, such analyses are complicated by the high mutation rates of RNA viruses, which can produce sequence similarities in distant strains by convergent mutations. By applying a genome-wide approach that examines the source of individual polymorphisms and that can be tested against null models in which recombination is absent and homoplasies can arise only by convergent mutations, we examine the extent and limits of recombination in Betacoronaviruses. We find that recombination accounts for nearly 40% of the polymorphisms circulating in populations and that gene exchange occurs almost exclusively among strains belonging to the same subgenus. Although experimental studies have shown that recombinational exchanges occur at random along the coronaviral genome, in nature, they are vastly overrepresented in regions controlling viral interaction with host cells.


Assuntos
Betacoronavirus/classificação , Betacoronavirus/genética , Recombinação Genética/genética , Glicoproteína da Espícula de Coronavírus/genética , Troca Genética/genética , Genes Virais/genética , Genoma Viral/genética , Especificidade de Hospedeiro/genética , Modelos Genéticos , Polimorfismo Genético , /genética , Tropismo Viral/genética
5.
Nat Commun ; 11(1): 6421, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33339818

RESUMO

Sexual reproduction is almost ubiquitous among extant eukaryotes. As most asexual lineages are short-lived, abandoning sex is commonly regarded as an evolutionary dead end. Still, putative anciently asexual lineages challenge this view. One of the most striking examples are bdelloid rotifers, microscopic freshwater invertebrates believed to have completely abandoned sexual reproduction tens of Myr ago. Here, we compare whole genomes of 11 wild-caught individuals of the bdelloid rotifer Adineta vaga and present evidence that some patterns in its genetic variation are incompatible with strict clonality and lack of genetic exchange. These patterns include genotype proportions close to Hardy-Weinberg expectations within loci, lack of linkage disequilibrium between distant loci, incongruent haplotype phylogenies across the genome, and evidence for hybridization between divergent lineages. Analysis of triallelic sites independently corroborates these findings. Our results provide evidence for interindividual genetic exchange and recombination in A. vaga, a species previously thought to be anciently asexual.


Assuntos
Genoma , Recombinação Genética/genética , Rotíferos/genética , Alelos , Animais , Genética Populacional , Células Germinativas/metabolismo , Haplótipos/genética , Desequilíbrio de Ligação/genética , Filogenia , Sequenciamento Completo do Genoma
6.
Anticancer Res ; 40(11): 6319-6325, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109569

RESUMO

BACKGROUND: Attempts have been made to enhance systemic therapy for osteosarcoma. In our previous study, the systemic administration of a vesicular stomatitis virus (VSV) improved the survival rates of mice with osteosarcoma but did not improve the long-term survival of the animals. MATERIALS AND METHODS: In the present study, we developed a novel oncolytic VSV by incorporating tumor-suppressor microRNA143 (rVSV-miR143) to compare the antitumor effects of various doses (10×10-4, 5×10-4, and 1×10-4 multiplicity of infection) of rVSV-miR143 with those of VSV in vitro. RESULTS: The cytotoxicity and migration-inhibitory effects of rVSV-miR143 on the osteosarcoma cells were significantly higher than those of VSV alone at a dose of 5×10-4 multiplicity of infection, indicating that rVSV-miRNA143 enhances the antitumor effect at certain doses. CONCLUSION: VSV incorporating tumor-suppressor miRNA143 demonstrated a synergistic antitumor effect on osteosarcoma cells in vitro.


Assuntos
Genes Supressores de Tumor , MicroRNAs/genética , Vírus Oncolíticos/genética , Recombinação Genética/genética , Vírus da Estomatite Vesicular Indiana/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , MicroRNAs/metabolismo , Terapia Viral Oncolítica , Osteossarcoma/genética , Osteossarcoma/patologia
7.
Proc Natl Acad Sci U S A ; 117(37): 22953-22961, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868446

RESUMO

The DNA-dependent protein kinase (DNA-PK), which is composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical nonhomologous end-joining (cNHEJ) factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends and recruits and activates DNA-PK. Activated DNA-PK phosphorylates DNA-PKcs at the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity but whether T2609 phosphorylation has a role in physiological DNA repair remains elusive. Using the DNA-PKcs 5A mouse model carrying alanine substitutions at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs 5A/5A B cells reveal increased chromosomal translocations, extensive use of distal switch regions (consistent with end resection), and preferential usage of microhomology-all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR.


Assuntos
Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Switching de Imunoglobulina/genética , Animais , Linfócitos B/imunologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Rearranjo Gênico , Humanos , Switching de Imunoglobulina/fisiologia , Região de Troca de Imunoglobulinas/genética , Imunoglobulinas/genética , Autoantígeno Ku/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Fosforilação , Recombinação Genética/genética , Translocação Genética
8.
Mol Immunol ; 127: 112-123, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32961421

RESUMO

A highly diverse repertoire of T cell antigen receptors (TCR) is created in the thymus by recombination of gene segments and the insertion or deletion of nucleotides at the junctions. Using next-generation TCR sequencing we define here the features of recombination and selection in the human TCRα and TCRß locus, and show that a strikingly high proportion of the repertoire is shared by unrelated individuals. The thymic TCRα nucleotide repertoire was more diverse than TCRß, with 4.1 × 106 vs. 0.81 × 106 unique clonotypes, and contained nonproductive clonotypes at a higher frequency (69.2% vs. 21.2%). The convergence of distinct nucleotide clonotypes to the same amino acid sequences was higher in TCRα than in TCRß repertoire (1.45 vs. 1.06 nucleotide sequences per amino acid sequence in thymus). The gene segment usage was biased, and generally all individuals favored the same genes in both TCRα and TCRß loci. Despite the high diversity, a large fraction of the repertoire was found in more than one donor. The shared fraction was bigger in TCRα than TCRß repertoire, and more common in in-frame sequences than in nonproductive sequences. Thus, both biases in rearrangement and thymic selection are likely to contribute to the generation of shared repertoire in humans.


Assuntos
Impressão Genômica , Linfócitos T/imunologia , Timo/citologia , Sequência de Bases , Células Clonais , Regiões Determinantes de Complementaridade/genética , Feminino , Variação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Mutagênese Insercional , Probabilidade , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Recombinação Genética/genética
9.
J Biosci Bioeng ; 130(4): 367-373, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32646632

RESUMO

Cross hybridization breeding of sake yeasts is hampered by difficulty in acquisition of haploid cells through sporulation. We previously demonstrated that typical sake yeast strains were defective in meiotic chromosome recombination, which caused poor sporulation and loss of spore viability. In this study, we screened a single copy plasmid genomic DNA library of the laboratory Saccharomyces cerevisiae GRF88 for genes that might complement the meiotic recombination defect of UTCAH-3, a strain derived from the sake yeast Kyokai no. 7 (K7). We identified the SPO11 gene of the laboratory strain (ScSPO11), encoding a meiosis-specific endonuclease that catalyzes DNA double-strand breaks required for meiotic recombination, as a gene that restored meiotic recombination and spore viability of UTCAH-3. K7SPO11 could not restore sporulation efficiency and spore viability of UTCAH-3 and a laboratory strain BY4743 spo11Δ/spo11Δ, indicating that K7SPO11 is not functional. Sequence analysis of the SPO11 genes of various Kyokai sake yeasts (K1, and K3-K10) revealed that the K7 group of sake yeasts (K6, K7, K9, and K10) had a mutual missense mutation (C73T) in addition to other three common mutations present in all Kyokai yeasts tested. ScSPO11C73T created through in vitro mutagenesis could not restore spore viability of BY4743 spo11Δ/spo11Δ. On the other hand, K8SPO11, which have the three common mutations except for C73T could restore spore viability of BY4743 spo11Δ/spo11Δ. These results suggest that C73T might be a causative mutation of recombination defect in K7SPO11. Moreover, we found that the introduction of ScRIM15 restored sporulation efficiency but not spore viability.


Assuntos
Bebidas Alcoólicas/microbiologia , Endodesoxirribonucleases/genética , Meiose/genética , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Clonagem Molecular , Quebras de DNA de Cadeia Dupla , Mutação , Saccharomyces cerevisiae/citologia
10.
BMC Bioinformatics ; 21(Suppl 12): 305, 2020 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-32703190

RESUMO

BACKGROUND: Horizontal gene transfer, i.e. the acquisition of genetic material from nonparent organism, is considered an important force driving species evolution. Many cases of horizontal gene transfer from prokaryotes to eukaryotes have been registered, but no transfer mechanism has been deciphered so far, although viruses were proposed as possible vectors in several studies. In agreement with this idea, in our previous study we discovered that in two eukaryotic proteins bacteriophage recombination site (AttP) was adjacent to the regions originating via horizontal gene transfer. In one of those cases AttP site was present inside the introns of cysteine-rich repeats. In the present study we aimed to apply computational tools for finding multiple horizontal gene transfer events in large genome databases. For that purpose we used a sequence of cysteine-rich repeats to identify genes potentially acquired through horizontal transfer. RESULTS: HMMER remote similarity search significantly detected 382 proteins containing cysteine-rich repeats. All of them, except 8 sequences, belong to eukaryotes. In 124 proteins the presence of conserved structural domains was predicted. In spite of the fact that cysteine-rich repeats are found almost exclusively in eukaryotic proteins, many predicted domains are most common for prokaryotes or bacteriophages. Ninety-eight proteins out of 124 contain typical prokaryotic domains. In those cases proteins were considered as potentially originating via horizontal transfer. In addition, HHblits search revealed that two domains of the same fungal protein, Glycoside hydrolase and Peptidase M15, have high similarity with proteins of two different prokaryotic species, hinting at independent horizontal gene transfer events. CONCLUSIONS: Cysteine-rich repeats in eukaryotic proteins are usually accompanied by conserved domains typical for prokaryotes or bacteriophages. These proteins, containing both cysteine-rich repeats, and characteristic prokaryotic domains, might represent multiple independent horizontal gene transfer events from prokaryotes to eukaryotes. We believe that the presence of bacteriophage recombination site inside cysteine-rich repeat coding sequence may facilitate horizontal genes transfer. Thus computational approach, described in the present study, can help finding multiple sequences originated from horizontal transfer in eukaryotic genomes.


Assuntos
Bacteriófagos/genética , Transferência Genética Horizontal/genética , Genes Virais , Recombinação Genética/genética , Proteínas Virais/química , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , Domínios Proteicos , Proteínas Virais/classificação
11.
Nucleic Acids Res ; 48(15): 8445-8460, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32644157

RESUMO

DNA lesions or other barriers frequently compromise replisome progress. The SF2 helicase RecG is a key enzyme in the processing of postreplication gaps or regressed forks in Escherichia coli. A deletion of the recG gene renders cells highly sensitive to a range of DNA damaging agents. Here, we demonstrate that RecG function is at least partially complemented by another SF2 helicase, RadD. A ΔrecGΔradD double mutant exhibits an almost complete growth defect, even in the absence of stress. Suppressors appear quickly, primarily mutations that compromise priA helicase function or recA promoter mutations that reduce recA expression. Deletions of uup (encoding the UvrA-like ABC system Uup), recO, or recF also suppress the ΔrecGΔradD growth phenotype. RadD and RecG appear to avoid toxic situations in DNA metabolism, either resolving or preventing the appearance of DNA repair intermediates produced by RecA or RecA-independent template switching at stalled forks or postreplication gaps. Barriers to replisome progress that require intervention by RadD or RecG occur in virtually every replication cycle. The results highlight the importance of the RadD protein for general chromosome maintenance and repair. They also implicate Uup as a new modulator of RecG function.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Reparo do DNA/genética , Replicação do DNA/genética , Proteínas de Escherichia coli/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Mutação/genética , Recombinação Genética/genética
12.
Sci Rep ; 10(1): 9397, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32523028

RESUMO

Pathogenicity islands (PAIs) represent horizontally acquired chromosomal regions and encode their cognate integrase, which mediates chromosomal integration and excision of the island. These site-specific recombination reactions have to be tightly controlled to maintain genomic stability, and their directionality depends on accessory proteins. The integration host factor (IHF) and the factor for inversion stimulation (Fis) are often involved in recombinogenic complex formation and controlling the directionality of the recombination reaction. We investigated the role of the accessory host factors IHF and Fis in controlling the stability of six PAIs in uropathogenic Escherichia coli strain 536. By comparing the loss of individual PAIs in the presence or absence of IHF or Fis, we showed that IHF specifically stabilized PAI I536 and that in particular the IHFB subunit seems to be important for this function. We employed complex genetic studies to address the role of IHF in PAI I536-encoded integrase (IntI) expression. Based on different YFP-reporter constructs and electrophoretic mobility shift assays we demonstrated that IntI acts a strong repressor of its own synthesis, and that IHF binding to the intI promoter region reduces the probability of intI promoter activation. Our results extend the current knowledge of the role of IHF in controlling directionality of site specific recombination reactions and thus PAI stability.


Assuntos
Proteínas de Escherichia coli/genética , Ilhas Genômicas/genética , Integrases/genética , Fatores Hospedeiros de Integração/genética , Regiões Promotoras Genéticas/genética , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/patogenicidade , Fator Proteico para Inversão de Estimulação/genética , Regulação Bacteriana da Expressão Gênica/genética , Recombinação Genética/genética
13.
Mol Cell Biol ; 40(16)2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32513818

RESUMO

Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) class switch recombination (CSR), somatic hypermutation (SHM), and gene conversion by converting DNA cytosines to uracils at specific genomic regions. In this study, we examined AID footprints across the entire length of an engineered switch region in cells ablated for uracil repair. We found that AID deamination occurs predominantly at WRC hot spots (where W is A or T and R is A or G) and that the deamination frequency remains constant across the entire switch region. Importantly, we analyzed monoallelic AID deamination footprints on both DNA strands occurring within a single cell cycle. We found that AID generates few and mostly isolated uracils in the switch region, although processive AID deaminations are evident in some molecules. The frequency of molecules containing deamination on both DNA strands at the acceptor switch region correlates with the class switch efficiency, raising the possibility that the minimal requirement for DNA double-strand break (DSB) formation is as low as even one AID deamination event on both DNA strands.


Assuntos
Linfócitos B/citologia , Citosina/metabolismo , Switching de Imunoglobulina/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Animais , Citidina Desaminase/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Desaminação/imunologia , Recombinação Genética/genética
14.
Am J Hum Genet ; 107(1): 137-148, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32533945

RESUMO

Recombination rates vary significantly across the genome, and estimates of recombination rates are needed for downstream analyses such as haplotype phasing and genotype imputation. Existing methods for recombination rate estimation are limited by insufficient amounts of informative genetic data or by high computational cost. We present a method and software, called IBDrecomb, for using segments of identity by descent to infer recombination rates. IBDrecomb can be applied to sequenced population cohorts to obtain high-resolution, population-specific recombination maps. In simulated admixed data, IBDrecomb obtains higher accuracy than admixture-based estimation of recombination rates. When applied to 2,500 simulated individuals, IBDrecomb obtains similar accuracy to a linkage-disequilibrium (LD)-based method applied to 96 individuals (the largest number for which computation is tractable). Compared to LD-based maps, our IBD-based maps have the advantage of estimating recombination rates in the recent past rather than the distant past. We used IBDrecomb to generate new recombination maps for European Americans and for African Americans from TOPMed sequence data from the Framingham Heart Study (1,626 unrelated individuals) and the Jackson Heart Study (2,046 unrelated individuals), and we compare them to LD-based, admixture-based, and family-based maps.


Assuntos
Recombinação Genética/genética , Afro-Americanos/genética , Grupo com Ancestrais do Continente Europeu/genética , Genética Populacional/métodos , Genoma Humano/genética , Haplótipos/genética , Humanos , Desequilíbrio de Ligação/genética , Polimorfismo de Nucleotídeo Único/genética
15.
Nat Commun ; 11(1): 3088, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32555206

RESUMO

DNA double-strand break repair by homologous recombination begins with nucleolytic resection of the 5' DNA strand at the break ends. Long-range resection is catalyzed by EXO1 and BLM-DNA2, which likely have to navigate through ribonucleotides and damaged bases. Here, we show that a short stretch of ribonucleotides at the 5' terminus stimulates resection by EXO1. Ribonucleotides within a 5' flap are resistant to cleavage by DNA2, and extended RNA:DNA hybrids inhibit both strand separation by BLM and resection by EXO1. Moreover, 8-oxo-guanine impedes EXO1 but enhances resection by BLM-DNA2, and an apurinic/apyrimidinic site stimulates resection by BLM-DNA2 and DNA strand unwinding by BLM. Accordingly, depletion of OGG1 or APE1 leads to greater dependence of DNA resection on DNA2. Importantly, RNase H2A deficiency impairs resection overall, which we attribute to the accumulation of long RNA:DNA hybrids at DNA ends. Our results help explain why eukaryotic cells possess multiple resection nucleases.


Assuntos
Quebras de DNA de Cadeia Dupla , Ribonucleotídeos/genética , Ribonucleotídeos/metabolismo , Western Blotting , Linhagem Celular Tumoral , DNA Glicosilases/genética , Enzimas Reparadoras do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Exodesoxirribonucleases/genética , Imunofluorescência , Recombinação Homóloga/genética , Humanos , RecQ Helicases/genética , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
16.
J Vis Exp ; (158)2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32364550

RESUMO

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian and avian host species, including humans. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing full-length rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral open reading frames, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.


Assuntos
Genes Reporter , Recombinação Genética/genética , Genética Reversa/métodos , Rotavirus/genética , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , RNA Polimerases Dirigidas por DNA/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Viral/genética , Análise de Sequência de RNA
17.
Nat Commun ; 11(1): 2141, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358538

RESUMO

Optogenetic genome engineering tools enable spatiotemporal control of gene expression and provide new insight into biological function. Here, we report the new version of genetically encoded photoactivatable (PA) Cre recombinase, PA-Cre 3.0. To improve PA-Cre technology, we compare light-dimerization tools and optimize for mammalian expression using a CAG promoter, Magnets, and 2A self-cleaving peptide. To prevent background recombination caused by the high sequence similarity in the dimerization domains, we modify the codons for mouse gene targeting and viral production. Overall, these modifications significantly reduce dark leak activity and improve blue-light induction developing our new version, PA-Cre 3.0. As a resource, we have generated and validated AAV-PA-Cre 3.0 as well as two mouse lines that can conditionally express PA-Cre 3.0. Together these new tools will facilitate further biological and biomedical research.


Assuntos
Integrases/metabolismo , Recombinação Genética/genética , Animais , Códon/genética , Engenharia Genética/métodos , Integrases/genética , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Optogenética , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/efeitos da radiação , Recombinação Genética/efeitos da radiação
18.
Viruses ; 12(5)2020 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-32370153

RESUMO

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a lethal zoonotic pathogen circulating in the Arabian Peninsula since 2012. There is no vaccine for MERS and anti-viral treatment is generally not applicable. We investigated the evolution of the MERS-CoV spike gene sequences and changes in viral loads over time from patients in Saudi Arabia from 2105-2017. All the MERS-CoV strains belonged to lineage 5, and showed high sequence homology (99.9%) to 2017 strains. Recombination analysis showed a potential recombination event in study strains from patients in Saudi Arabia. The spike gene showed eight amino acid substitutions, especially between the A1 and B5 lineage, and contained positively selected codon 1020. We also determined that the viral loads were significantly (p < 0.001) higher in fatal cases, and virus shedding was prolonged in some fatal cases beyond 21 days. The viral concentration peaked during the first week of illness, and the lower respiratory specimens had higher levels of MERS-CoV RNA. The presence of the diversifying selection and the topologies with the structural mapping of residues under purifying selection suggested that codon 1020 might have a role in the evolution of spike gene during the divergence of different lineages. This study will im-prove our understanding of the evolution of MERS-CoV, and also highlights the need for enhanced surveillance in humans and dromedaries. The presence of amino acid changes at the N-terminal domain and structural mapping of residues under positive selection at heptad repeat 1 provides better insight into the adaptive evolution of the spike gene and might have a potential role in virus-host tropism and pathogenesis.


Assuntos
Substituição de Aminoácidos/genética , Infecções por Coronavirus/patologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Glicoproteína da Espícula de Coronavírus/genética , Adulto , Idoso , Sequência de Aminoácidos , Animais , Sequência de Bases , Camelus/virologia , Dipeptidil Peptidase 4/metabolismo , Evolução Molecular , Feminino , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Domínios Proteicos/genética , RNA Viral/genética , Receptores Virais/genética , Recombinação Genética/genética , Arábia Saudita , Análise de Sequência de RNA , Homologia de Sequência , Carga Viral , Tropismo Viral/genética
19.
Mol Phylogenet Evol ; 149: 106848, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32380283

RESUMO

Sugarcane mosaic virus (SCMV), a member of the genus Potyvirus in the family Potyviridae, is an important pathogen that causes mosaic diseases in maize, sugarcane, canna and other graminaceous species worldwide. Previously, several reports have showed the genetic variation and population structure of SCMV. However, the evolutionary dynamics, synonymous codon usage pattern and adaptive evolution of the virus is unclear. In this study, we performed comprehensive analyses of phylodynamics, composition bias and codon usage of SCMV using 108 complete genomic sequences. Our phylogenetic analysis found six host- and geographically confined phylogenetic lineages within the SCMV non-recombinant isolates. We found a relatively stable and conserved genomic composition with a lower codon usage choice in the SCMV protein coding sequences. Mutation pressure and natural selection have shaped the codon usage patterns of the SCMV protein coding sequences with natural selection being the dominant factor. The codon adaptation index (CAI), relative codon deoptimization index (RCDI) and similarity index (SiD) analyses revealed a stronger correlation between SCMV and maize than between SCMV and sugarcane or canna. Our study is the first to evaluate the codon usage pattern of SCMV based on complete sequences and may provide a better understanding of the origin of SCMV and its evolutionary patterns for future research.


Assuntos
Adaptação Fisiológica/genética , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Potyvirus/genética , Sequência de Bases , Teorema de Bayes , Códon/genética , Funções Verossimilhança , Nucleotídeos/genética , Fases de Leitura Aberta/genética , Filogenia , Análise de Componente Principal , Recombinação Genética/genética
20.
Nature ; 582(7812): 426-431, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32461690

RESUMO

Sex chromosomes in males of most eutherian mammals share only a small homologous segment, the pseudoautosomal region (PAR), in which the formation of double-strand breaks (DSBs), pairing and crossing over must occur for correct meiotic segregation1,2. How cells ensure that recombination occurs in the PAR is unknown. Here we present a dynamic ultrastructure of the PAR and identify controlling cis- and trans-acting factors that make the PAR the hottest segment for DSB formation in the male mouse genome. Before break formation, multiple DSB-promoting factors hyperaccumulate in the PAR, its chromosome axes elongate and the sister chromatids separate. These processes are linked to heterochromatic mo-2 minisatellite arrays, and require MEI4 and ANKRD31 proteins but not the axis components REC8 or HORMAD1. We propose that the repetitive DNA sequence of the PAR confers unique chromatin and higher-order structures that are crucial for recombination. Chromosome synapsis triggers collapse of the elongated PAR structure and, notably, oocytes can be reprogrammed to exhibit spermatocyte-like levels of DSBs in the PAR simply by delaying or preventing synapsis. Thus, the sexually dimorphic behaviour of the PAR is in part a result of kinetic differences between the sexes in a race between the maturation of the PAR structure, formation of DSBs and completion of pairing and synapsis. Our findings establish a mechanistic paradigm for the recombination of sex chromosomes during meiosis.


Assuntos
Quebras de DNA de Cadeia Dupla , Meiose , Regiões Pseudoautossômicas/genética , Regiões Pseudoautossômicas/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Pareamento Cromossômico/genética , Proteínas de Ligação a DNA , Feminino , Heterocromatina/genética , Heterocromatina/metabolismo , Heterocromatina/ultraestrutura , Cinética , Masculino , Meiose/genética , Camundongos , Repetições Minissatélites/genética , Oócitos/metabolismo , Recombinação Genética/genética , Caracteres Sexuais , Troca de Cromátide Irmã , Espermatócitos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
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