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1.
Proc Natl Acad Sci U S A ; 117(40): 24947-24956, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32968016

RESUMO

The acquisition of mutations plays critical roles in adaptation, evolution, senescence, and tumorigenesis. Massive genome sequencing has allowed extraction of specific features of many mutational landscapes but it remains difficult to retrospectively determine the mechanistic origin(s), selective forces, and trajectories of transient or persistent mutations and genome rearrangements. Here, we conducted a prospective reciprocal approach to inactivate 13 single or multiple evolutionary conserved genes involved in distinct genome maintenance processes and characterize de novo mutations in 274 diploid Saccharomyces cerevisiae mutation accumulation lines. This approach revealed the diversity, complexity, and ultimate uniqueness of mutational landscapes, differently composed of base substitutions, small insertions/deletions (InDels), structural variants, and/or ploidy variations. Several landscapes parallel the repertoire of mutational signatures in human cancers while others are either novel or composites of subsignatures resulting from distinct DNA damage lesions. Notably, the increase of base substitutions in the homologous recombination-deficient Rad51 mutant, specifically dependent on the Polζ translesion polymerase, yields COSMIC signature 3 observed in BRCA1/BRCA2-mutant breast cancer tumors. Furthermore, "mutome" analyses in highly polymorphic diploids and single-cell bottleneck lineages revealed a diverse spectrum of loss-of-heterozygosity (LOH) signatures characterized by interstitial and terminal chromosomal events resulting from interhomolog mitotic cross-overs. Following the appearance of heterozygous mutations, the strong stimulation of LOHs in the rad27/FEN1 and tsa1/PRDX1 backgrounds leads to fixation of homozygous mutations or their loss along the lineage. Overall, these mutomes and their trajectories provide a mechanistic framework to understand the origin and dynamics of genome variations that accumulate during clonal evolution.


Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Mutação/genética , Saccharomyces cerevisiae/genética , Acetiltransferases/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/patologia , Dano ao DNA/genética , DNA Polimerase Dirigida por DNA , Diploide , Feminino , Endonucleases Flap/genética , Genoma Fúngico/genética , Humanos , Perda de Heterozigosidade/genética , Proteínas de Membrana/genética , Peroxirredoxinas/genética , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
2.
Nat Commun ; 11(1): 4212, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32839469

RESUMO

Phosphatases, together with kinases and transcription factors, are key components in cellular signalling networks. Here, we present a systematic functional analysis of the phosphatases in Cryptococcus neoformans, a fungal pathogen that causes life-threatening fungal meningoencephalitis. We analyse 230 signature-tagged mutant strains for 114 putative phosphatases under 30 distinct in vitro growth conditions, revealing at least one function for 60 of these proteins. Large-scale virulence and infectivity assays using insect and mouse models indicate roles in pathogenicity for 31 phosphatases involved in various processes such as thermotolerance, melanin and capsule production, stress responses, O-mannosylation, or retromer function. Notably, phosphatases Xpp1, Ssu72, Siw14, and Sit4 promote blood-brain barrier adhesion and crossing by C. neoformans. Together with our previous systematic studies of transcription factors and kinases, our results provide comprehensive insight into the pathobiological signalling circuitry of C. neoformans.


Assuntos
Cryptococcus neoformans/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica/métodos , Genoma Fúngico/genética , Estudo de Associação Genômica Ampla/métodos , Monoéster Fosfórico Hidrolases/genética , Animais , Análise por Conglomerados , Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Feminino , Proteínas Fúngicas/classificação , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Camundongos Endogâmicos , Monoéster Fosfórico Hidrolases/classificação , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/classificação , Fosfotransferases/genética , Fosfotransferases/metabolismo , Transdução de Sinais/genética , Termotolerância/genética , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência/genética
3.
F1000Res ; 92020.
Artigo em Inglês | MEDLINE | ID: mdl-32765832

RESUMO

Diversity within the fungal kingdom is evident from the wide range of morphologies fungi display as well as the various ecological roles and industrial purposes they serve. Technological advances, particularly in long-read sequencing, coupled with the increasing efficiency and decreasing costs across sequencing platforms have enabled robust characterization of fungal genomes. These sequencing efforts continue to reveal the rampant diversity in fungi at the genome level. Here, we discuss studies that have furthered our understanding of fungal genetic diversity and genomic evolution. These studies revealed the presence of both small-scale and large-scale genomic changes. In fungi, research has recently focused on many small-scale changes, such as how hypermutation and allelic transmission impact genome evolution as well as how and why a few specific genomic regions are more susceptible to rapid evolution than others. High-throughput sequencing of a diverse set of fungal genomes has also illuminated the frequency, mechanisms, and impacts of large-scale changes, which include chromosome structural variation and changes in chromosome number, such as aneuploidy, polyploidy, and the presence of supernumerary chromosomes. The studies discussed herein have provided great insight into how the architecture of the fungal genome varies within species and across the kingdom and how modern fungi may have evolved from the last common fungal ancestor and might also pave the way for understanding how genomic diversity has evolved in all domains of life.


Assuntos
Fungos , Genoma Fúngico , Fungos/genética , Genoma Fúngico/genética , Genômica
4.
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
5.
Proc Natl Acad Sci U S A ; 117(31): 18608-18616, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690696

RESUMO

Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that removes RNA polymerase (RNAP)-stalling DNA damage from the transcribed strand (TS) of active genes. TC-NER deficiency in humans is associated with the severe neurological disorder Cockayne syndrome. Initiation of TC-NER is mediated by specific factors such as the human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of the chromatin organization, is unclear. Here, we used single-nucleotide resolution UV damage mapping data to show that Rad26 and its ATPase activity is critical for TC-NER downstream of the first (+1) nucleosome in gene coding regions. However, TC-NER on the transcription start site (TSS)-proximal half of the +1 nucleosome is largely independent of Rad26, likely due to high occupancy of the transcription initiation/repair factor TFIIH in this nucleosome. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER in Rad26-deficient cells. We show that deletion of SPT4 significantly restores TC-NER across the genome in a rad26∆ mutant, particularly in the downstream nucleosomes. These data demonstrate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5 in transcribed chromatin and Rad26 mainly functions downstream of the +1 nucleosome to remove TC-NER suppression by Spt4/Spt5.


Assuntos
Adenosina Trifosfatases , Reparo do DNA/genética , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA Helicases , Enzimas Reparadoras do DNA , Genoma Fúngico/genética , Humanos , Nucleossomos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Nature ; 582(7810): 119-123, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494069

RESUMO

The three-dimensional architecture of the genome governs its maintenance, expression and transmission. The cohesin protein complex organizes the genome by topologically linking distant loci, and is highly enriched in specialized chromosomal domains surrounding centromeres, called pericentromeres1-6. Here we report the three-dimensional structure of pericentromeres in budding yeast (Saccharomyces cerevisiae) and establish the relationship between genome organization and function. We find that convergent genes mark pericentromere borders and, together with core centromeres, define their structure and function by positioning cohesin. Centromeres load cohesin, and convergent genes at pericentromere borders trap it. Each side of the pericentromere is organized into a looped conformation, with border convergent genes at the base. Microtubule attachment extends a single pericentromere loop, size-limited by convergent genes at its borders. Reorienting genes at borders into a tandem configuration repositions cohesin, enlarges the pericentromere and impairs chromosome biorientation during mitosis. Thus, the linear arrangement of transcriptional units together with targeted cohesin loading shapes pericentromeres into a structure that is competent for chromosome segregation. Our results reveal the architecture of the chromosomal region within which kinetochores are embedded, as well as the restructuring caused by microtubule attachment. Furthermore, we establish a direct, causal relationship between the three-dimensional genome organization of a specific chromosomal domain and cellular function.


Assuntos
Centrômero/genética , Centrômero/metabolismo , Genes Fúngicos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/metabolismo , Centrômero/química , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Genoma Fúngico/genética , Viabilidade Microbiana/genética , Mitose/genética , Conformação Molecular
7.
Arch Microbiol ; 202(8): 2033-2058, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32506150

RESUMO

Polyaromatic hydrocarbons (PAHs) are considered as hazardous organic priority pollutants. PAHs have immense public concern and critical environmental challenge around the globe due to their toxic, carcinogenic, and mutagenic properties, and their ubiquitous distribution, recalcitrance as well as persistence in environment. The knowledge about harmful effects of PAHs on ecosystem along with human health has resulted in an interest of researchers on degradation of these compounds. Whereas physico-chemical treatment of PAHs is cost and energy prohibitive, bioremediation i.e. degradation of PAHs using microbes is becoming an efficient and sustainable approach. Broad range of microbes including bacteria, fungi, and algae have been found to have capability to use PAHs as carbon and energy source under both aerobic and anaerobic conditions resulting in their transformation/degradation. Microbial genetic makeup containing genes encoding catabolic enzymes is responsible for PAH-degradation mechanism. The degradation capacity of microbes may be induced by exposing them to higher PAH-concentration, resulting in genetic adaptation or changes responsible for high efficiency towards removal/degradation. In last few decades, mechanism of PAH-biodegradation, catabolic gene system encoding catabolic enzymes, and genetic adaptation and regulation have been investigated in detail. This review is an attempt to overview current knowledge of microbial degradation mechanism of PAHs, its genetic regulation with application of genetic engineering to construct genetically engineered microorganisms, specific catabolic enzyme activity, and application of bioremediation for reclamation of PAH-contaminated sites. In addition, advanced molecular techniques i.e. genomic, proteomic, and metabolomic techniques are also discussed as powerful tools for elucidation of PAH-biodegradation/biotransformation mechanism in an environmental matrix.


Assuntos
Bactérias , Biodegradação Ambiental , Microbiologia Ambiental , Poluentes Ambientais/metabolismo , Fungos , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Bactérias/genética , Bactérias/metabolismo , Biotransformação , Fungos/genética , Fungos/metabolismo , Engenharia Genética , Genoma Bacteriano/genética , Genoma Fúngico/genética
8.
Nat Commun ; 11(1): 2144, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358542

RESUMO

The Saccharomycotina subphylum (budding yeasts) spans 400 million years of evolution and includes species that thrive in diverse environments. To study niche-adaptation, we identify changes in gene expression in three divergent yeasts grown in the presence of various stressors. Duplicated and non-conserved genes are significantly more likely to respond to stress than genes that are conserved as single-copy orthologs. Next, we develop a sorting method that considers evolutionary origin and duplication timing to assign an evolutionary age to each gene. Subsequent analysis reveals that genes that emerged in recent evolutionary time are enriched amongst stress-responsive genes for each species. This gene expression pattern suggests that budding yeasts share a stress adaptation mechanism, whereby selective pressure leads to functionalization of young genes to improve growth in adverse conditions. Further characterization of young genes from species that thrive in harsh environments can inform the design of more robust strains for biotechnology.


Assuntos
Saccharomycetales/metabolismo , Ascomicetos/genética , Ascomicetos/metabolismo , Biotecnologia/métodos , Genoma Fúngico/genética , Filogenia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
PLoS One ; 15(5): e0227396, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469865

RESUMO

Elsinoë fawcettii, a necrotrophic fungal pathogen, causes citrus scab on numerous citrus varieties around the world. Known pathotypes of E. fawcettii are based on host range; additionally, cryptic pathotypes have been reported and more novel pathotypes are thought to exist. E. fawcettii produces elsinochrome, a non-host selective toxin which contributes to virulence. However, the mechanisms involved in potential pathogen-host interactions occurring prior to the production of elsinochrome are unknown, yet the host-specificity observed among pathotypes suggests a reliance upon such mechanisms. In this study we have generated a whole genome sequencing project for E. fawcettii, producing an annotated draft assembly 26.01 Mb in size, with 10,080 predicted gene models and low (0.37%) coverage of transposable elements. A small proportion of the assembly showed evidence of AT-rich regions, potentially indicating genomic regions with increased plasticity. Using a variety of computational tools, we mined the E. fawcettii genome for potential virulence genes as candidates for future investigation. A total of 1,280 secreted proteins and 276 candidate effectors were predicted and compared to those of other necrotrophic (Botrytis cinerea, Parastagonospora nodorum, Pyrenophora tritici-repentis, Sclerotinia sclerotiorum and Zymoseptoria tritici), hemibiotrophic (Leptosphaeria maculans, Magnaporthe oryzae, Rhynchosporium commune and Verticillium dahliae) and biotrophic (Ustilago maydis) plant pathogens. Genomic and proteomic features of known fungal effectors were analysed and used to guide the prioritisation of 120 candidate effectors of E. fawcettii. Additionally, 378 carbohydrate-active enzymes were predicted and analysed for likely secretion and sequence similarity with known virulence genes. Furthermore, secondary metabolite prediction indicated nine additional genes potentially involved in the elsinochrome biosynthesis gene cluster than previously described. A further 21 secondary metabolite clusters were predicted, some with similarity to known toxin producing gene clusters. The candidate virulence genes predicted in this study provide a comprehensive resource for future experimental investigation into the pathogenesis of E. fawcettii.


Assuntos
Ascomicetos/genética , Citrus/microbiologia , Genoma Fúngico/genética , Anotação de Sequência Molecular , Ascomicetos/patogenicidade , Parede Celular/enzimologia , Mineração de Dados , Família Multigênica/genética , Metabolismo Secundário/genética
10.
BMC Evol Biol ; 20(1): 58, 2020 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-32448114

RESUMO

BACKGROUND: Functional constraint through genomic architecture is suggested to be an important dimension of genome evolution, but quantitative evidence for this idea is rare. In this contribution, existing evidence and discussions on genomic architecture as constraint for convergent evolution, rapid adaptation, and genic adaptation are summarized into alternative, testable hypotheses. Network architecture statistics from protein-protein interaction networks are then used to calculate differences in evolutionary outcomes on the example of genomic evolution in yeast, and the results are used to evaluate statistical support for these longstanding hypotheses. RESULTS: A discriminant function analysis lent statistical support to classifying the yeast interactome into hub, intermediate and peripheral nodes based on network neighborhood connectivity, betweenness centrality, and average shortest path length. Quantitative support for the existence of genomic architecture as a mechanistic basis for evolutionary constraint is then revealed through utilizing these statistical parameters of the protein-protein interaction network in combination with estimators of protein evolution. CONCLUSIONS: As functional genetic networks are becoming increasingly available, it will now be possible to evaluate functional genetic network constraint against variables describing complex phenotypes and environments, for better understanding of commonly observed deterministic patterns of evolution in non-model organisms. The hypothesis framework and methodological approach outlined herein may help to quantify the extrinsic versus intrinsic dimensions of evolutionary constraint, and result in a better understanding of how fast, effectively, or deterministically organisms adapt.


Assuntos
Evolução Molecular , Redes Reguladoras de Genes , Genoma Fúngico/genética , Fenótipo , Saccharomyces cerevisiae/genética
11.
Nucleic Acids Res ; 48(11): 5799-5813, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32399566

RESUMO

Transcription and pre-mRNA splicing are coupled to promote gene expression and regulation. However, mechanisms by which transcription and splicing influence each other are still under investigation. The ATPase Prp5p is required for pre-spliceosome assembly and splicing proofreading at the branch-point region. From an open UV mutagenesis screen for genetic suppressors of prp5 defects and subsequent targeted testing, we identify components of the TBP-binding module of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, Spt8p and Spt3p. Spt8Δ and spt3Δ rescue the cold-sensitivity of prp5-GAR allele, and prp5 mutants restore growth of spt8Δ and spt3Δ strains on 6-azauracil. By chromatin immunoprecipitation (ChIP), we find that prp5 alleles decrease recruitment of RNA polymerase II (Pol II) to an intron-containing gene, which is rescued by spt8Δ. Further ChIP-seq reveals that global effects on Pol II-binding are mutually rescued by prp5-GAR and spt8Δ. Inhibited splicing caused by prp5-GAR is also restored by spt8Δ. In vitro assays indicate that Prp5p directly interacts with Spt8p, but not Spt3p. We demonstrate that Prp5p's splicing proofreading is modulated by Spt8p and Spt3p. Therefore, this study reveals that interactions between the TBP-binding module of SAGA and the spliceosomal ATPase Prp5p mediate a balance between transcription initiation/elongation and pre-spliceosome assembly.


Assuntos
RNA Helicases DEAD-box/metabolismo , Processamento de RNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Alelos , Genes Fúngicos/genética , Genoma Fúngico/genética , Mutação , Fenótipo , Ligação Proteica , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
12.
Sci Rep ; 10(1): 5884, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32246017

RESUMO

Heterobasidion irregulare and H. occidentale are two closely related conifer root rot pathogens in the H. annosum sensu lato (s.l.) species complex. The two species H. irregulare and H. occidentale have different host preference with pine and non-pine tree species favored, respectively. The comparison of transcriptomes of H. irregulare and H. occidentale growing in Norway spruce bark, a susceptible host non-native to North America, showed large differences in gene expression. Heterobasidion irregulare induced more genes involved in detoxification of host compounds and in production of secondary metabolites, while the transcriptome induced in H. occidentale was more oriented towards carbohydrate degradation. Along with their separated evolutionary history, the difference might be driven by their host preferences as indicated by the differentially expressed genes enriched in particular Gene Ontology terms.


Assuntos
Basidiomycota/fisiologia , Picea/microbiologia , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , Basidiomycota/genética , Basidiomycota/patogenicidade , Regulação da Expressão Gênica de Plantas , Genoma Fúngico/genética , Picea/metabolismo , Casca de Planta/metabolismo , Virulência
13.
Gene ; 742: 144586, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32179171

RESUMO

Pycnoporus sanguineus, an edible mushroom, produces antimicrobial and antitumor bioactive compounds and pH- and thermo- stable laccases that have multiple potential biotechnological applications. Here we reported the complete genome of the species Pycnoporus sanguineus ACCC 51,180 by using the combination of Illumina HiSeq X Ten and the PacBio sequencing technology. The represented genome is 36.6 Mb composed of 59 scaffolds with 12,086 functionally annotated protein-coding genes. The genome of Pycnoporus sanguineus encodes at least 19 biosynthetic gene clusters for secondary metabolites, including a terpene cluster for biosynthesis of the antitumor clavaric acid. Seven laccases were identified, while 22 genes were found to be involved in the kynurenine pathway in which the intermediate metabolite 3-hydroxyanthranilic acid were catalyzed by laccases into cinnabarinic acid. This study represented the third genome of the genus Pycnoporus, and wound facilitate the exploration of useful sources from Pycnoporus sanguineus for future industrial applications.


Assuntos
Proteínas Fúngicas/genética , Genoma Fúngico/genética , Microbiologia Industrial/métodos , Lacase/genética , Pycnoporus/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Cinurenina/metabolismo , Lacase/metabolismo , Engenharia Metabólica , Oxazinas/metabolismo , Estabilidade Proteica , Pycnoporus/enzimologia , Metabolismo Secundário/genética
14.
Parasitol Res ; 119(5): 1433-1441, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32200463

RESUMO

The reduction and specialization of the energy metabolism system is a common trait in the evolution of intracellular parasites. One group of fungi-related parasites, the Microsporidia, seems to have developed this trait far more than other eukaryotes. As an extreme adaptation for a parasitic lifestyle, some of them have completely lost the ability to synthesize ATP, relying heavily upon the metabolic processes of host cells to ensure their own development and reproduction. For a long time, only fragmentary data on the functioning and evolution of the energy metabolism system in microsporidia was available. However, the recent discovery of microsporidia-related microorganisms, the Cryptomycota and Aphelida, alongside with the genome sequencing and new data about basal groups in the Microsporidia has shed new light on this problem. Here, we review recent data about functioning of the energy metabolism system in microsporidia and closely related organisms, and discuss possible evolutionary pathways in the group.


Assuntos
Metabolismo Energético/genética , Evolução Molecular , Microsporídios/genética , Microsporídios/metabolismo , Fungos/classificação , Fungos/genética , Fungos/metabolismo , Genoma Fúngico/genética , Interações Hospedeiro-Parasita , Mutação com Perda de Função , Microsporídios/classificação , Filogenia
15.
Proc Natl Acad Sci U S A ; 117(14): 7917-7928, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32193338

RESUMO

A fundamental characteristic of eukaryotic organisms is the generation of genetic variation via sexual reproduction. Conversely, significant large-scale genome structure variations could hamper sexual reproduction, causing reproductive isolation and promoting speciation. The underlying processes behind large-scale genome rearrangements are not well understood and include chromosome translocations involving centromeres. Recent genomic studies in the Cryptococcus species complex revealed that chromosome translocations generated via centromere recombination have reshaped the genomes of different species. In this study, multiple DNA double-strand breaks (DSBs) were generated via the CRISPR/Cas9 system at centromere-specific retrotransposons in the human fungal pathogen Cryptococcus neoformans The resulting DSBs were repaired in a complex manner, leading to the formation of multiple interchromosomal rearrangements and new telomeres, similar to chromothripsis-like events. The newly generated strains harboring chromosome translocations exhibited normal vegetative growth but failed to undergo successful sexual reproduction with the parental wild-type strain. One of these strains failed to produce any spores, while another produced ∼3% viable progeny. The germinated progeny exhibited aneuploidy for multiple chromosomes and showed improved fertility with both parents. All chromosome translocation events were accompanied without any detectable change in gene sequences and thus suggest that chromosomal translocations alone may play an underappreciated role in the onset of reproductive isolation and speciation.


Assuntos
Centrômero/genética , Criptococose/genética , Cryptococcus neoformans/genética , Isolamento Reprodutivo , Sistemas CRISPR-Cas/genética , Instabilidade Cromossômica/genética , Cromossomos/genética , Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Quebras de DNA de Cadeia Dupla , Genoma Fúngico/genética , Genômica , Humanos , Translocação Genética/genética
16.
Gene ; 741: 144559, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-32169630

RESUMO

The fungi in order Mortierellales are attractive producers for long-chain polyunsaturated fatty acids (PUFAs). Here, the genome sequencing and assembly of a novel strain of Mortierella sp. BCC40632 were done, yielding 65 contigs spanning of 49,964,116 total bases with predicted 12,149 protein-coding genes. We focused on the acetyl-CoA in relevant to its derived metabolic pathways for biosynthesis of macromolecules with biological functions, including PUFAs, eicosanoids and carotenoids. By comparative genome analysis between Mortierellales and Mucorales, the signature genetic characteristics of the arachidonic acid-producing strains, including Δ5-desaturase and GLELO-like elongase, were also identified in the strain BCC40632. Remarkably, this fungal strain contained only n-6 pathway of PUFA biosynthesis due to the absence of Δ15-desaturase or ω3-desaturase gene in contrast to other Mortierella species. Four putative enzyme sequences in the eicosanoid biosynthetic pathways were identified in the strain BCC40632 and others Mortierellale fungi, but were not detected in the Mucorales. Another unique metabolic trait of the Mortierellales was the inability in carotenoid synthesis as a result of the lack of phytoene synthase and phytoene desaturase genes. The findings provide a perspective in strain optimization for production of tailored-made products with industrial applications.


Assuntos
Acetilcoenzima A/biossíntese , Ácido Araquidônico/genética , Genoma Fúngico/genética , Mortierella/metabolismo , Acetilcoenzima A/genética , Ácido Araquidônico/biossíntese , Vias Biossintéticas/genética , Ácidos Graxos Dessaturases/genética , Elongases de Ácidos Graxos/genética , Ácidos Graxos Insaturados/genética , Ácidos Graxos Insaturados/metabolismo , Mortierella/genética , Mucorales/genética , Mucorales/metabolismo , Ácido gama-Linolênico/genética , Ácido gama-Linolênico/metabolismo
17.
Mol Plant Microbe Interact ; 33(4): 576-579, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32013763

RESUMO

Elsinoë ampelina is an ascomycetous fungus that causes grape anthracnose, a potentially devastating disease worldwide. Here, we report a 28.29 Mb high-quality genome sequence of E. ampelina YL-1 that encodes 8,057 predicted protein-coding genes and represents the first sequenced genome assembly of E. ampelina. This study adds to the current genomic resources for the genus Elsinoë and paves the way for research on comparative genomic studies, E. ampelina-grape interactions, and improvement of management strategies.


Assuntos
Ascomicetos , Genoma Fúngico , Vitis , Ascomicetos/genética , Genoma Fúngico/genética , Genômica , Doenças das Plantas/microbiologia , Vitis/microbiologia
18.
Nucleic Acids Res ; 48(6): 3053-3070, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32020204

RESUMO

The S phase checkpoint is crucial to maintain genome stability under conditions that threaten DNA replication. One of its critical functions is to prevent Exo1-dependent fork degradation, and Exo1 is phosphorylated in response to different genotoxic agents. Exo1 seemed to be regulated by several post-translational modifications in the presence of replicative stress, but the specific contribution of checkpoint-dependent phosphorylation to Exo1 control and fork stability is not clear. We show here that Exo1 phosphorylation is Dun1-independent and Rad53-dependent in response to DNA damage or dNTP depletion, and in both situations Exo1 is similarly phosphorylated at multiple sites. To investigate the correlation between Exo1 phosphorylation and fork stability, we have generated phospho-mimic exo1 alleles that rescue fork collapse in rad53 mutants as efficiently as exo1-nuclease dead mutants or the absence of Exo1, arguing that Rad53-dependent phosphorylation is the mayor requirement to preserve fork stability. We have also shown that this rescue is Bmh1-2 independent, arguing that the 14-3-3 proteins are dispensable for fork stabilization, at least when Exo1 is downregulated. Importantly, our results indicated that phosphorylation specifically inhibits the 5' to 3'exo-nuclease activity, suggesting that this activity of Exo1 and not the flap-endonuclease, is the enzymatic activity responsible of the collapse of stalled replication forks in checkpoint mutants.


Assuntos
Proteínas 14-3-3/genética , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Exodesoxirribonucleases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Ciclo Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Genoma Fúngico/genética , Instabilidade Genômica/genética , Fosforilação/genética , Processamento de Proteína Pós-Traducional/genética , Pontos de Checagem da Fase S do Ciclo Celular/genética , Saccharomyces cerevisiae/genética
19.
Nat Commun ; 11(1): 809, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32041946

RESUMO

Transcription-replication conflicts (TRCs) occur when intensive transcriptional activity compromises replication fork stability, potentially leading to gene mutations. Transcription-deposited H3K4 methylation (H3K4me) is associated with regions that are susceptible to TRCs; however, the interplay between H3K4me and TRCs is unknown. Here we show that H3K4me aggravates TRC-induced replication failure in checkpoint-defective cells, and the presence of methylated H3K4 slows down ongoing replication. Both S-phase checkpoint activity and H3K4me are crucial for faithful DNA synthesis under replication stress, especially in highly transcribed regions where the presence of H3K4me is highest and TRCs most often occur. H3K4me mitigates TRCs by decelerating ongoing replication, analogous to how speed bumps slow down cars. These findings establish the concept that H3K4me defines the transcriptional status of a genomic region and defends the genome from TRC-mediated replication stress and instability.


Assuntos
Replicação do DNA , Histonas/metabolismo , Transcrição Genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Cromatina/metabolismo , DNA Polimerase II/metabolismo , Genoma Fúngico/genética , Instabilidade Genômica , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Metilação , Modelos Genéticos , Mutação , Pontos de Checagem da Fase S do Ciclo Celular/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Appl Microbiol Biotechnol ; 104(7): 3037-3047, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32043190

RESUMO

Targeted gene mutation by allelic replacement is important for functional genomic analysis and metabolic engineering. However, it is challenging in mutating the essential genes with the traditional method by using a selection marker, since the first step of essential gene knockout will result in a lethal phenotype. Here, we developed a two-end selection marker (Two-ESM) method for site-directed mutation of essential genes in Saccharomyces cerevisiae with the aid of the CRISPR/Cas9 system. With this method, single and double mutations of the essential gene ERG20 (encoding farnesyl diphosphate synthase) in S. cerevisiae were successfully constructed with high efficiencies of 100%. In addition, the Two-ESM method significantly improved the mutation efficiency and simplified the genetic manipulation procedure compared with traditional methods. The genome integration and mutation efficiencies were further improved by dynamic regulation of mutant gene expression and optimization of the integration modules. This Two-ESM method will facilitate the construction of genomic mutations of essential genes for functional genomic analysis and metabolic flux regulation in yeasts. KEY POINTS: • A Two-ESM strategy achieves mutations of essential genes with high efficiency of 100%. • The optimized three-module method improves the integration efficiency by more than three times. • This method will facilitate the functional genomic analysis and metabolic flux regulation.


Assuntos
Genes Essenciais/genética , Genoma Fúngico/genética , Mutagênese Sítio-Dirigida , Saccharomyces cerevisiae/genética , Sistemas CRISPR-Cas , Edição de Genes , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Marcadores Genéticos/genética , Genômica , Engenharia Metabólica , Mutação , Saccharomyces cerevisiae/metabolismo
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