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1.
Sheng Wu Gong Cheng Xue Bao ; 35(9): 1761-1770, 2019 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-31559757

RESUMO

Seamless modification is a popular genomic manipulation technique in genetic engineering. Selection stringency of the counter-selection system determines the efficiency of the seamless modification. Recently, a novel counter-selection system, kil, was constructed. It is reported that the selection selectivity of kil is higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, indicating that recombinants could be selected out more efficiently by combining kil counter-selection system and plasmid pSim6. In order to confirm this speculation, four different loci (lacI, dbpa, ack, glk) in Escherichia coli strains W3110, MG1655 and DH10B were selected for testing: dsDNA fragments of different sizes (500 bp, 1 000 bp, and 2 000 bp) were used to substitute tet/kil. As expected, recombination efficiency was higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, and the results were more obvious with the length of dsDNA increasing. Specifically, recombination efficiency was 1.2 to 2 fold higher in pSim6 harboring bacteria than in pKD46 harboring bacteria when dsDNA fragments were 1 000 bp in length. With the length of dsDNA increasing up to 2 000 bp, the gap increased to 2.2-5 fold. In conclusion, it is easier to perform seamless modification by combining kil counter-selection system and plasmid pSim6 than combining kil and pKD46. An alternative tool in genomic engineering is provided in this study.


Assuntos
Engenharia Genética , Escherichia coli , Proteínas de Escherichia coli , Plasmídeos , Recombinação Genética
4.
Zhonghua Yu Fang Yi Xue Za Zhi ; 53(8): 811-816, 2019 Aug 06.
Artigo em Chinês | MEDLINE | ID: mdl-31378041

RESUMO

Objective: To analyze the infection status and recombination of Norovirus in patients with acute gastroenteritis in Ningxia. Methods: The specimens of 10 sentinel hospitals in Ningxia were collected from 2016 to 2017. Real-time quantitative PCR was used for nucleic acid detection. GⅡ-positive samples were amplified by RT-PCR for the RdRp and Capsid regions, then sequenced and genotyped. Evolution analysis was performed using software such as MEGA-X, and recombination analysis was performed using Simplot 3.5.1 and RDP4. Results: The age of the 2 334 cases was 1.42 (0.68, 7.69) years old, 1 133 cases in 2016 and 1 201 cases in 2017, 1 343 and 991 cases for males and females respectively. The positive rate of Norovirus GⅠ genogroup was 0.86% (20/2 334), and GⅡ genogroup was 14.82% (346/2 334). A total of 78 recombinant strains were sequenced and 12 recombinant types were found. GⅡ.Pe/GⅡ.4Sydney_2012 and GⅡ.P12/GⅡ.3 were the main epidemic strains, accounting for 35.90% (28 strains) and 32.05% (25 strain) respectively, followed by GⅡ.P16/GⅡ.2 accounting for 12.82% (10 strains). Among them,GⅡ.P7/GⅡ.6 (2 strains), GⅡ.P12/GⅡ.3 (6 strains), GⅡ.P16/GⅡ.1 (2 strains), GⅡ.P16/GⅡ.2 (5 strains), GⅡ.Pe/GⅡ.4 (7 strains) were detected for the first time in Ningxia. Recombinant strains were all intergenotype recombination, and the recombination breakpionts were all located within ORF1. Conclusion: Norovirus infection in Ningxia area was mainly in GⅡ genogroup from 2016 to 2017, and most of them were recombinant strains. GⅡ.Pe/GⅡ.4Sydney_2012 and GⅡ.P12/GⅡ.3 were the main epidemic strains, followed by GⅡ.P16/GⅡ. 2.


Assuntos
Infecções por Caliciviridae/epidemiologia , Gastroenterite/virologia , Norovirus/genética , Recombinação Genética , Criança , China , Feminino , Gastroenterite/epidemiologia , Genótipo , Humanos , Masculino , Filogenia
5.
Arch Virol ; 164(10): 2605-2608, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31300889

RESUMO

Porcine reproductive and respiratory syndrome virus 1 is a major cause of swine morbidity and mortality in various parts of the world, including Hungary. A national elimination programme to reduce the associated economic burden was initiated in Hungary in 2012. Using extensive laboratory surveillance, we identified and isolated an unusual PRRSV strain. The complete coding sequence of this isolate was determined and analyzed. The genome of this Hungarian PRRSV1 strain, HUN60077/16, is 15,081 nucleotides in length. Phylogenetic and recombination analysis showed a mosaic structure of the genome where a large fragment of ORF1b and the genomic region coding for ORF3 to ORF7 showed a very close genetic relationship to the vaccine virus Unistrain, while the ORF1a region, the 3' end of ORF1b, and the whole ORF2 were only distantly related to this or any other PRRSV1 strain whose genome sequence is available in the GenBank database. Genomic characterization of PRRSV strains is crucial when possible vaccine-associated cases are identified. This approach not only helps to identify genetic interactions between vaccine and wild-type PRRSV1 strains but may also be needed to prevent trust in commercial vaccines from being undermined.


Assuntos
Genoma Viral , Filogenia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Recombinação Genética , Vacinas Virais/genética , Animais , Genótipo , Hungria , Fases de Leitura Aberta , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Análise de Sequência de DNA , Homologia de Sequência , Suínos
6.
BMC Plant Biol ; 19(1): 310, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307401

RESUMO

BACKGROUND: The hypersensitive defense response (HR) in plants is a fast, localized necrotic response around the point of pathogen ingress. HR is usually triggered by a pathogen recognition event mediated by a nucleotide-binding site, leucine-rich repeat (NLR) protein. The autoactive maize NLR gene Rp1-D21 confers a spontaneous HR response in the absence of pathogen recognition. Previous work identified a set of loci associated with variation in the strength of Rp1-D21-induced HR. A polygalacturonase gene homolog, here termed ZmPGH1, was identified as a possible causal gene at one of these loci on chromosome 7. RESULTS: Expression of ZmPGH1 inhibited the HR-inducing activity of both Rp1-D21 and that of another autoactive NLR, RPM1(D505V), in a Nicotiana benthamiana transient expression assay system. Overexpression of ZmPGH1 in a transposon insertion line of maize was associated with suppression of chemically-induced programmed cell death and with suppression of HR induced by Rp1-D21 in maize plants grown in the field. CONCLUSIONS: ZmPGH1 functions as a suppressor of programmed cell death induced by at least two autoactive NLR proteins and by two chemical inducers. These findings deepen our understanding of the control of the HR in plants.


Assuntos
Apoptose/fisiologia , Proteínas de Plantas/fisiologia , Poligalacturonase/fisiologia , Zea mays/fisiologia , Apoptose/genética , Mapeamento Cromossômico , Cromossomos de Plantas , Genes de Plantas , Leucina , Proteínas de Plantas/química , Proteínas de Plantas/genética , Poligalacturonase/química , Poligalacturonase/genética , Recombinação Genética , Sequências Repetitivas de Aminoácidos , Tabaco/genética , Zea mays/enzimologia , Zea mays/genética , Zea mays/imunologia
7.
Arch Virol ; 164(10): 2551-2558, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31321588

RESUMO

Here, we report two novel enteroviruses, designated as SD-S67 and SD-S68, isolated from a goat farm. Their complete genome sequences were determined and found to be 7455 and 7465 nucleotides in length, respectively. Molecular characterization revealed that SD-S67 is closely related to bovine enterovirus strain 261 and that SD-S68 to caprine enterovirus strain CEV-JL14. Phylogenetic analysis showed that SD-S67 clustered with members of the species Enterovirus F, and that SD-S68 clustered with enteroviruses of goats and sheep. Recombination analysis showed that SD-S67 is likely to have undergone several recombination events in the process of its evolution. To the best of our knowledge, this is the first report of an enterovirus F isolate from a goat and of a coinfection with enteroviruses of different species in the same goat herd.


Assuntos
Infecções por Enterovirus/veterinária , Enterovirus/classificação , Enterovirus/isolamento & purificação , Doenças das Cabras/virologia , Filogenia , Animais , Análise por Conglomerados , Efeito Citopatogênico Viral , Enterovirus/genética , Infecções por Enterovirus/virologia , Genoma Viral , Cabras , Microscopia Eletrônica de Transmissão , Recombinação Genética , Análise de Sequência de DNA , Homologia de Sequência , Vírion/ultraestrutura , Cultura de Vírus
8.
Plant Dis ; 103(9): 2451-2459, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322491

RESUMO

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most devastating wheat diseases in Ethiopia. To study virulence genetics of the pathogen, 117 progeny isolates were produced through sexual reproduction of an Ethiopian isolate of the stripe rust pathogen on Berberis holstii plants under controlled conditions. The parental and progeny isolates were characterized by phenotyping on wheat lines carrying single Yr genes for resistance and genotyped using 10 polymorphic simple sequence repeated (SSR) markers. The progeny isolates were classified into 37 virulence phenotypes and 75 multilocus genotypes. The parental isolate and progeny isolates were all avirulent to resistance genes Yr5, Yr10, Yr15, Yr24, Yr32, YrTr1, YrSP, and Yr76 but virulent to Yr1 and Yr2, indicating that the parental isolate was homozygous avirulent or homozygous virulent at these loci. The progeny isolates segregated for virulence to 12 Yr genes. Virulence phenotypes to Yr6, Yr28, Yr43, and Yr44 were controlled by a single dominant gene; those to Yr7, Yr9, Yr17, Yr27, Yr25, Yr31, and YrExp2 were each controlled by two dominant genes; and the virulence phenotype to Yr8 was controlled by two complementary dominant genes. A linkage map was constructed with seven SSR markers, and 16 virulence loci corresponding to 11 Yr resistance genes were mapped with some loci linked to each other. These results are useful in understanding host-pathogen interactions and selecting resistance genes to develop wheat cultivars with highly effective resistance to stripe rust.


Assuntos
Basidiomycota , Berberis , Ligação Genética , Recombinação Genética , Virulência , Basidiomycota/genética , Basidiomycota/patogenicidade , Berberis/genética , Etiópia , Doenças das Plantas , Triticum/microbiologia , Virulência/genética
9.
Nat Commun ; 10(1): 2472, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31171781

RESUMO

The evolution of microbial and viral organisms often generates clonal interference, a mode of competition between genetic clades within a population. Here we show how interference impacts systems biology by constraining genetic and phenotypic complexity. Our analysis uses biophysically grounded evolutionary models for molecular phenotypes, such as fold stability and enzymatic activity of genes. We find a generic mode of phenotypic interference that couples the function of individual genes and the population's global evolutionary dynamics. Biological implications of phenotypic interference include rapid collateral system degradation in adaptation experiments and long-term selection against genome complexity: each additional gene carries a cost proportional to the total number of genes. Recombination above a threshold rate can eliminate this cost, which establishes a universal, biophysically grounded scenario for the evolution of sex. In a broader context, our analysis suggests that the systems biology of microbes is strongly intertwined with their mode of evolution.


Assuntos
Bactérias/genética , Evolução Biológica , Dobramento de Proteína , Estabilidade Proteica , Vírus/genética , Bactérias/metabolismo , Evolução Molecular , Aptidão Genética , Fenótipo , Recombinação Genética , Seleção Genética , Biologia de Sistemas , Vírus/metabolismo
10.
Arch Virol ; 164(9): 2379-2383, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31203434

RESUMO

During 2017, leaf samples of chili pepper (Capsicum annuum), tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum) plants exhibiting yellowing and curling symptoms were collected from Aceh province, Indonesia. These samples were used to isolate and sequence viral genomic DNA. Six isolates with complete DNA-A and DNA-B sequences of begomovirus were obtained, all of which showed >99% sequence identity to the others. DNA-A sequences shared the highest nucleotide sequence identity (89.3%-89.7%) with monopartite pepper yellow leaf curl Indonesia virus 2 (PepYLCIV2) and the second-highest sequence identity (87.3%-87.4%) with bipartite pepper yellow leaf curl Indonesia virus (PepYLCIV). The DNA-B sequences shared the highest nucleotide sequence identity (95%-97.5%) with PepYLCIV. Results of recombination analysis indicated that the novel begomovirus was a recombinant. In accordance with the guidelines for begomovirus species demarcation, these isolates should be assigned to a new species, and we have proposed the name ''pepper yellow leaf curl Aceh virus'' (PepYLCAV) for this virus.


Assuntos
Begomovirus/isolamento & purificação , Capsicum/virologia , Lycopersicon esculentum/virologia , Doenças das Plantas/virologia , Tabaco/virologia , Begomovirus/classificação , Begomovirus/genética , Genoma Viral , Indonésia , Filogenia , Recombinação Genética , Análise de Sequência de DNA
11.
Nat Commun ; 10(1): 2862, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253793

RESUMO

DNA double strand breaks (DSBs) pose a high risk for genome integrity. Cells repair DSBs through homologous recombination (HR) when a sister chromatid is available. HR is upregulated by the cycling dependent kinase (CDK) despite the paradox of telophase, where CDK is high but a sister chromatid is not nearby. Here we study in the budding yeast the response to DSBs in telophase, and find they activate the DNA damage checkpoint (DDC), leading to a telophase-to-G1 delay. Outstandingly, we observe a partial reversion of sister chromatid segregation, which includes approximation of segregated material, de novo formation of anaphase bridges, and coalescence between sister loci. We finally show that DSBs promote a massive change in the dynamics of telophase microtubules (MTs), together with dephosphorylation and relocalization of kinesin-5 Cin8. We propose that chromosome segregation is not irreversible and that DSB repair using the sister chromatid is possible in telophase.


Assuntos
Cromátides/metabolismo , Segregação de Cromossomos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Fúngico/genética , Troca de Cromátide Irmã , Telófase/genética , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Food Chem Toxicol ; 131: 110557, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31176925

RESUMO

The aim of the present study was to appraise the mutagenic and recombinogenic potential of bupropion hydrochloride (BHc) and trazodone hydrochloride (THc). We used standard (ST) and the high bioactivation (HB) crossings from Drosophila melanogaster in the Somatic Mutation and Recombination Test. We treated third-instar larvae from both crossings with different concentrations of BHc and THc (0.9375 to 7.5 mg/mL). BHc significantly increased the frequency of mutant spots in both crossings, except for the lowest concentration in the ST crossing. ST had also the mostly recombinogenic result, and in the HB, BHc was highly mutagenic. On the other hand, THc significantly increased the frequency of mutant spots in both the ST and HB crossings at all concentrations. The three initial concentrations were recombinogenic and the highest concentration was mutagenic for the THc. BHc and THc at high concentrations were toxic, even though their mutagenicity was not dose-related. THc significantly increased the frequency of mutant spots when metabolized, probably as a result of the production of 1-(3'-chlorophenyl) piperazine. BHc was essentially recombinogenic and when metabolized, it became mutagenic. THc was recombinogenic in both crossings. Further studies are needed to clarify the action mechanisms from BHc and THc.


Assuntos
Antidepressivos/toxicidade , Bupropiona/toxicidade , Drosophila melanogaster/efeitos dos fármacos , Mutagênicos/toxicidade , Recombinação Genética/efeitos dos fármacos , Trazodona/toxicidade , Animais , Drosophila melanogaster/genética , Feminino , Masculino , Testes de Mutagenicidade , Mutação , Asas de Animais/efeitos dos fármacos
13.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 827-836, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31223001

RESUMO

Exonuclease Ⅷ (Exo Ⅷ), an ATP-independent dsDNA 5'-3' exonuclease, is a candidate protein with great application value for in vitro DNA recombination. However, the application of Exo Ⅷ in DNA recombination in vitro has not been reported. In this study, the recombinant expression vector of the truncated Exo Ⅷ (tExo Ⅷ) with the full exonuclease activity was built and used to achieve the overexpression of tExo Ⅷ in Escherichia coli. Based on the purified tExo Ⅷ protein with high-purity, the feasibility of tExo Ⅷ applied in vitro DNA recombination and effects of the reaction temperatures, reaction duration, and homology arm lengths were examined. The results showed that tExo Ⅷ was highly expressed in soluble form in E. coli. One liter of bacterial culture yielded 92.40 mg of purified tExo Ⅷ with the specific activity of 1.21×105 U/mg. In a 10 µL recombination system containing 2.5 U tExo Ⅷ, the highest cloning efficiency was achieved in a reaction at 25 °C for 12.5 min and followed by incubation at 50 °C for 50 min. With addition of Pfu DNA polymerase, the homology arm extension strategy can effectively improve the recombination efficiency. Using competent E. coli Mach1 T1 with 2.2×106 cfu/µg transformation efficiency as recipient cell, the recombination of a 1 kb fragment with a 21 bp homology arm and a 5.8 kb linearized vector can form about 1.1×104 recombinant clones per µg vector, and the positive rates was over 80%. The recombination efficiency was increased with the increasing length of homology arm ranged from 8 to 21 bp. Under the optimal reaction condition, only 8 bp homology arm can still achieve valid DNA recombination. This novel in vitro DNA recombination system mediated by tExo Ⅷ was particularly characterized by its easy preparation, no limitation on restriction sites and high recombination cloning efficiency. All results revealed that the new efficient gene cloning system has potential application in the field of molecular biology.


Assuntos
Exonucleases , Proteínas Recombinantes , Recombinação Genética , Clonagem Molecular , Escherichia coli/genética , Exonucleases/genética , Proteínas Recombinantes/metabolismo
14.
Genome Biol Evol ; 11(1): 1780-1796, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31173069

RESUMO

The diversification of microbial populations may be driven by many factors including adaptation to distinct ecological niches and barriers to recombination. We examined the population structure of the bacterial pathogen Pseudomonas aeruginosa by analyzing whole-genome sequences of 739 isolates from diverse sources. We confirmed that the population structure of P. aeruginosa consists of two major groups (referred to as Groups A and B) and at least two minor groups (Groups C1 and C2). Evidence for frequent intragroup but limited intergroup recombination in the core genome was observed, consistent with sexual isolation of the groups. Likewise, accessory genome analysis demonstrated more gene flow within Groups A and B than between these groups, and a few accessory genomic elements were nearly specific to one or the other group. In particular, the exoS gene was highly overrepresented in Group A compared with Group B isolates (99.4% vs. 1.1%) and the exoU gene was highly overrepresented in Group B compared with Group A isolates (95.2% vs. 1.8%). The exoS and exoU genes encode effector proteins secreted by the P. aeruginosa type III secretion system. Together these results suggest that the major P. aeruginosa groups defined in part by the exoS and exoU genes are divergent from each other, and that these groups are genetically isolated and may be ecologically distinct. Although both groups were globally distributed and caused human infections, certain groups predominated in some clinical contexts.


Assuntos
ADP Ribose Transferases/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Genética Populacional , Pseudomonas aeruginosa/genética , Fluxo Gênico , Genoma Bacteriano , Filogenia , Recombinação Genética
15.
Malar J ; 18(1): 150, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035999

RESUMO

BACKGROUND: The high proportion of human cases due to the simian malaria parasite Plasmodium knowlesi in Malaysia is a cause of concern, as they can be severe and even fatal. Merozoite surface protein 7 (MSP7) is a multigene family which forms a non-covalent complex with MSP-1 prior to receptor-ligand recognition in Plasmodium falciparum and thus an important antigen for vaccine development. However, no study has been done in any of the ortholog family members in P. knowlesi from clinical samples. This study investigates the level of polymorphism, haplotypes, and natural selection acting at the pkmsp-7D gene in clinical samples from Malaysia. METHODS: Thirty-six full-length pkmsp7D gene sequences (along with the reference H-strain: PKNH_1266000) obtained from clinical isolates of Malaysia, which were orthologous to pvmsp7H (PVX_082680) were downloaded from public databases. Population genetic, evolutionary and phylogenetic analyses were performed to determine the level of genetic diversity, polymorphism, recombination and natural selection. RESULTS: Analysis of 36 full-length pkmsp7D sequences identified 147 SNPs (91 non-synonymous and 56 synonymous substitutions). Nucleotide diversity across the full-length gene was higher than its ortholog in Plasmodium vivax (msp7H). Region-wise analysis of the gene indicated that the nucleotide diversity at the central region was very high (π = 0.14) compared to the 5' and 3' regions. Most hyper-variable SNPs were detected at the central domain. Multiple test for natural selection indicated the central region was under strong positive natural selection however, the 5' and 3' regions were under negative/purifying selection. Evidence of intragenic recombination were detected at the central region of the gene. Phylogenetic analysis using full-length msp7D genes indicated there was no geographical clustering of parasite population. CONCLUSIONS: High genetic diversity with hyper-variable SNPs and strong evidence of positive natural selection at the central region of MSP7D indicated exposure of the region to host immune pressure. Negative selection at the 5' and the 3' regions of MSP7D might be because of functional constraints at the unexposed regions during the merozoite invasion process of P. knowlesi. No evidence of geographical clustering among the clinical isolates from Malaysia indicated uniform selection pressure in all populations. These findings highlight the further evaluation of the regions and functional characterization of the protein as a potential blood stage vaccine candidate for P. knowlesi.


Assuntos
Variação Genética , Proteínas de Membrana/genética , Plasmodium knowlesi/genética , Proteínas de Protozoários/genética , Seleção Genética , Haplótipos , Humanos , Malária/parasitologia , Malásia , Filogenia , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Análise de Sequência de DNA
16.
BMC Plant Biol ; 19(1): 172, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31039740

RESUMO

BACKGROUND: Angiosperm sex chromosomes, where present, are generally recently evolved. The key step in initiating the development of sex chromosomes from autosomes is the establishment of a sex-determining locus within a region of non-recombination. To better understand early sex chromosome evolution, it is important to determine the process by which recombination is suppressed around the sex determining genes. We have used the dioecious angiosperm kiwifruit Actinidia chinensis var. chinensis, which has an active-Y sex chromosome system, to study recombination rates around the sex locus, to better understand key events in the development of sex chromosomes. RESULTS: We have confirmed the sex-determining region (SDR) in A. chinensis var. chinensis, using a combination of high density genetic mapping and fluorescent in situ hybridisation (FISH) of Bacterial Artificial Chromosomes (BACs) linked to the sex markers onto pachytene chromosomes. The SDR is a subtelomeric non-recombining region adjacent to the nucleolar organiser region (NOR). A region of restricted recombination of around 6 Mbp in size in both male and female maps spans the SDR and covers around a third of chromosome 25. CONCLUSIONS: As recombination is suppressed over a similar region between X chromosomes and between and X and Y chromosomes, we propose that recombination is suppressed in this region because of the proximity of the NOR and the centromere, with both the NOR and centromere suppressing recombination, and this predates suppressed recombination due to differences between X and Y chromosomes. Such regions of suppressed recombination in the genome provide an opportunity for the evolution of sex chromosomes, if a sex-determining locus develops there or translocates into this region.


Assuntos
Actinidia/genética , Cromossomos de Plantas , Recombinação Genética , Cromossomos Sexuais , Actinidia/citologia , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos , Variação Genética , Hibridização in Situ Fluorescente , Repetições de Microssatélites
17.
Nat Genet ; 51(5): 877-884, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31043755

RESUMO

Like many other crops, the cultivated peanut (Arachis hypogaea L.) is of hybrid origin and has a polyploid genome that contains essentially complete sets of chromosomes from two ancestral species. Here we report the genome sequence of peanut and show that after its polyploid origin, the genome has evolved through mobile-element activity, deletions and by the flow of genetic information between corresponding ancestral chromosomes (that is, homeologous recombination). Uniformity of patterns of homeologous recombination at the ends of chromosomes favors a single origin for cultivated peanut and its wild counterpart A. monticola. However, through much of the genome, homeologous recombination has created diversity. Using new polyploid hybrids made from the ancestral species, we show how this can generate phenotypic changes such as spontaneous changes in the color of the flowers. We suggest that diversity generated by these genetic mechanisms helped to favor the domestication of the polyploid A. hypogaea over other diploid Arachis species cultivated by humans.


Assuntos
Arachis/genética , Arachis/classificação , Argentina , Cromossomos de Plantas/genética , Produtos Agrícolas/genética , Metilação de DNA , DNA de Plantas/genética , Domesticação , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Variação Genética , Genoma de Planta , Hibridização Genética , Fenótipo , Poliploidia , Recombinação Genética , Especificidade da Espécie , Tetraploidia
18.
Nat Protoc ; 14(6): 1820-1840, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110297

RESUMO

Fate mapping is a powerful genetic tool for linking stem or progenitor cells with their progeny, and hence for defining cell lineages in vivo. The resolution of fate mapping depends on the numbers of distinct markers that are introduced in the beginning into stem or progenitor cells; ideally, numbers should be sufficiently large to allow the tracing of output from individual cells. Highly diverse genetic barcodes can serve this purpose. We recently developed an endogenous genetic barcoding system, termed Polylox. In Polylox, random DNA recombination can be induced by transient activity of Cre recombinase in a 2.1-kb-long artificial recombination substrate that has been introduced into a defined locus in mice (Rosa26Polylox reporter mice). Here, we provide a step-by-step protocol for the use of Polylox, including barcode induction and estimation of induction efficiency, barcode retrieval with single-molecule real-time (SMRT) DNA sequencing followed by computational barcode identification, and the calculation of barcode-generation probabilities, which is key for estimations of single-cell labeling for a given number of stem cells. Thus, Polylox barcoding enables high-resolution fate mapping in essentially all tissues in mice for which inducible Cre driver lines are available. Alternative methods include ex vivo cell barcoding, inducible transposon insertion and CRISPR-Cas9-based barcoding; Polylox currently allows combining non-invasive and cell-type-specific labeling with high label diversity. The execution time of this protocol is ~2-3 weeks for experimental data generation and typically <2 d for computational Polylox decoding and downstream analysis.


Assuntos
Linhagem da Célula , Genes Reporter , Análise de Sequência de DNA/métodos , Animais , Sistemas CRISPR-Cas , DNA/genética , DNA/metabolismo , Código de Barras de DNA Taxonômico/métodos , Feminino , Técnicas de Genotipagem/métodos , Integrases/genética , Masculino , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase/métodos , Recombinação Genética
19.
Nat Genet ; 51(6): 1035-1043, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31133745

RESUMO

Group A Streptococcus (GAS; Streptococcus pyogenes) is a bacterial pathogen for which a commercial vaccine for humans is not available. Employing the advantages of high-throughput DNA sequencing technology to vaccine design, we have analyzed 2,083 globally sampled GAS genomes. The global GAS population structure reveals extensive genomic heterogeneity driven by homologous recombination and overlaid with high levels of accessory gene plasticity. We identified the existence of more than 290 clinically associated genomic phylogroups across 22 countries, highlighting challenges in designing vaccines of global utility. To determine vaccine candidate coverage, we investigated all of the previously described GAS candidate antigens for gene carriage and gene sequence heterogeneity. Only 15 of 28 vaccine antigen candidates were found to have both low naturally occurring sequence variation and high (>99%) coverage across this diverse GAS population. This technological platform for vaccine coverage determination is equally applicable to prospective GAS vaccine antigens identified in future studies.


Assuntos
Genômica , Vacinas Estreptocócicas/genética , Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/imunologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Genoma Bacteriano , Estudo de Associação Genômica Ampla , Genômica/métodos , Humanos , Filogenia , Recombinação Genética , Infecções Estreptocócicas/prevenção & controle , Streptococcus pyogenes/classificação
20.
Virol J ; 16(1): 63, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068194

RESUMO

BACKGROUND: Hand, foot, and mouth disease (HFMD) is a common childhood disease, which is usually caused by enterovirus A (EV-A) serotypes. Enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are the main etiologic agents. Multiple serotypes of enterovirus B serotypes (EV-B) have been detected in outbreaks or sporadic cases of HFMD. RESULTS: During HFMD surveillance in Yunnan, China in 2013, two echovirus 33 (E-33) isolates were recovered in cell culture and typed by molecular methods from the cerebrospinal fluid (CSF) and feces of two sporadic cases of HFMD complicated by meningitis. Sequence analysis indicated that the study isolates, YNK35 and YNA12, formed an independent branch, and belonged to E-33 genotype H. Recombination analysis indicated multiple recombination events in the genomic sequence of isolate YNK35. The recombination mainly occurred in the non-structural coding region of P2 and P3, and involved intra-species recombination of species B. CONCLUSION: In this study, the complete sequences of two E-33 isolates were determined. This is the first report of severe HFMD associated with E-33 in Yunnan China, and it enriches the number of full-length genome sequences of E-33 in the GenBank database.


Assuntos
Enterovirus Humano B/genética , Doença de Mão, Pé e Boca/complicações , Doença de Mão, Pé e Boca/virologia , Meningite/virologia , Recombinação Genética , China/epidemiologia , Enterovirus Humano B/isolamento & purificação , Monitoramento Epidemiológico , Feminino , Variação Genética , Genoma Viral , Doença de Mão, Pé e Boca/líquido cefalorraquidiano , Humanos , Lactente , Masculino , Filogenia , Sorogrupo , Sequenciamento Completo do Genoma
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