RESUMO
Here we describe a competitive genome editing method that measures the effect of mutations on molecular functions, based on precision CRISPR editing using template libraries with either the original or altered sequence, and a sequence tag, enabling direct comparison between original and mutated cells. Using the example of the MYC oncogene, we identify important transcriptional targets and show that E-box mutations at MYC target gene promoters reduce cellular fitness.
Assuntos
Edição de Genes , Fatores de Transcrição , Sítios de Ligação/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The transcription factor Myc drives cell growth across animal phyla and is activated in most forms of human cancer. However, it is unclear which Myc target genes need to be regulated to induce growth and whether multiple targets act additively or if induction of each target is individually necessary. Here, we identified Myc target genes whose regulation is conserved between humans and flies and deleted Myc-binding sites (E-boxes) in the promoters of fourteen of these genes in Drosophila. E-box mutants of essential genes were homozygous viable, indicating that the E-boxes are not required for basal expression. Eight E-box mutations led to Myc-like phenotypes; the strongest mutant, ppanEbox-/-, also made the flies resistant to Myc-induced cell growth without affecting Myc-induced apoptosis. The ppanEbox-/- flies are healthy and display only a minor developmental delay, suggesting that it may be possible to treat or prevent tumorigenesis by targeting individual downstream targets of Myc.
Assuntos
Proteínas Proto-Oncogênicas c-myc , Ribossomos , Animais , Proliferação de Células/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ribossomos/metabolismo , Ativação Transcricional , Regulação para CimaRESUMO
DNA can determine where and when genes are expressed, but the full set of sequence determinants that control gene expression is unknown. Here, we measured the transcriptional activity of DNA sequences that represent an ~100 times larger sequence space than the human genome using massively parallel reporter assays (MPRAs). Machine learning models revealed that transcription factors (TFs) generally act in an additive manner with weak grammar and that most enhancers increase expression from a promoter by a mechanism that does not appear to involve specific TF-TF interactions. The enhancers themselves can be classified into three types: classical, closed chromatin and chromatin dependent. We also show that few TFs are strongly active in a cell, with most activities being similar between cell types. Individual TFs can have multiple gene regulatory activities, including chromatin opening and enhancing, promoting and determining transcription start site (TSS) activity, consistent with the view that the TF binding motif is the key atomic unit of gene expression.
Assuntos
Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição , Sítios de Ligação/genética , Genoma Humano/genética , Humanos , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
RNA-binding proteins (RBPs) regulate RNA metabolism at multiple levels by affecting splicing of nascent transcripts, RNA folding, base modification, transport, localization, translation, and stability. Despite their central role in RNA function, the RNA-binding specificities of most RBPs remain unknown or incompletely defined. To address this, we have assembled a genome-scale collection of RBPs and their RNA-binding domains (RBDs) and assessed their specificities using high-throughput RNA-SELEX (HTR-SELEX). Approximately 70% of RBPs for which we obtained a motif bound to short linear sequences, whereas â¼30% preferred structured motifs folding into stem-loops. We also found that many RBPs can bind to multiple distinctly different motifs. Analysis of the matches of the motifs in human genomic sequences suggested novel roles for many RBPs. We found that three cytoplasmic proteins-ZC3H12A, ZC3H12B, and ZC3H12C-bound to motifs resembling the splice donor sequence, suggesting that these proteins are involved in degradation of cytoplasmic viral and/or unspliced transcripts. Structural analysis revealed that the RNA motif was not bound by the conventional C3H1 RNA-binding domain of ZC3H12B. Instead, the RNA motif was bound by the ZC3H12B's PilT N terminus (PIN) RNase domain, revealing a potential mechanism by which unconventional RBDs containing active sites or molecule-binding pockets could interact with short, structured RNA molecules. Our collection containing 145 high-resolution binding specificity models for 86 RBPs is the largest systematic resource for the analysis of human RBPs and will greatly facilitate future analysis of the various biological roles of this important class of proteins.
Assuntos
Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/química , RNA/metabolismo , Sequência de Bases , Genoma Humano , Humanos , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Ribonucleases/química , Ribonucleases/metabolismo , Técnica de Seleção de AptâmerosRESUMO
Point mutations in cancer have been extensively studied but chromosomal gains and losses have been more challenging to interpret due to their unspecific nature. Here we examine high-resolution allelic imbalance (AI) landscape in 1699 colorectal cancers, 256 of which have been whole-genome sequenced (WGSed). The imbalances pinpoint 38 genes as plausible AI targets based on previous knowledge. Unbiased CRISPR-Cas9 knockout and activation screens identified in total 79 genes within AI peaks regulating cell growth. Genetic and functional data implicate loss of TP53 as a sufficient driver of AI. The WGS highlights an influence of copy number aberrations on the rate of detected somatic point mutations. Importantly, the data reveal several associations between AI target genes, suggesting a role for a network of lineage-determining transcription factors in colorectal tumorigenesis. Overall, the results unravel the contribution of AI in colorectal cancer and provide a plausible explanation why so few genes are commonly affected by point mutations in cancers.
Assuntos
Desequilíbrio Alélico , Neoplasias Colorretais/genética , Predisposição Genética para Doença , Sistemas CRISPR-Cas , Aberrações Cromossômicas , Cromossomos Humanos Par 8 , Neoplasias Colorretais/patologia , Variações do Número de Cópias de DNA , Dinamarca , Perfilação da Expressão Gênica , Genômica , Genótipo , Humanos , Perda de Heterozigosidade , Repetições de Microssatélites , Fenótipo , Mutação Puntual , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Interferente Pequeno/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Sequenciamento Completo do GenomaRESUMO
No existing method to characterize transcription factor (TF) binding to DNA allows genome-wide measurement of all TF-binding activity in cells. Here we present a massively parallel protein activity assay, active TF identification (ATI), that measures the DNA-binding activity of all TFs in cell or tissue extracts. ATI is based on electrophoretic separation of protein-bound DNA sequences from a highly complex DNA library and subsequent mass-spectrometric identification of the DNA-bound proteins. We applied ATI to four mouse tissues and mouse embryonic stem cells and found that, in a given tissue or cell type, a small set of TFs, which bound to only â¼10 distinct motifs, displayed strong DNA-binding activity. Some of these TFs were found in all cell types, whereas others were specific TFs known to determine cell fate in the analyzed tissue or cell type. We also show that a small number of TFs determined the accessible chromatin landscape of a cell, suggesting that gene regulatory logic may be simpler than previously appreciated.
Assuntos
Cromatina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Biotecnologia , Diferenciação Celular , Cromatina/genética , DNA/genética , DNA/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Especificidade da Espécie , Distribuição TecidualRESUMO
In some dimeric cases of transcription factor (TF) binding, the specificity of dimeric motifs has been observed to differ notably from what would be expected were the two factors to bind to DNA independently of each other. Current motif discovery methods are unable to learn monomeric and dimeric motifs in modular fashion such that deviations from the expected motif would become explicit and the noise from dimeric occurrences would not corrupt monomeric models. We propose a novel modeling technique and an expectation maximization algorithm, implemented as software tool MODER, for discovering monomeric TF binding motifs and their dimeric combinations. Given training data and seeds for monomeric motifs, the algorithm learns in the same probabilistic framework a mixture model which represents monomeric motifs as standard position-specific probability matrices (PPMs), and dimeric motifs as pairs of monomeric PPMs, with associated orientation and spacing preferences. For dimers the model represents deviations from pure modular model of two independent monomers, thus making co-operative binding effects explicit. MODER can analyze in reasonable time tens of Mbps of training data. We validated the tool on HT-SELEX and ChIP-seq data. Our findings include some TFs whose expected model has palindromic symmetry but the observed model is directional.
Assuntos
DNA/química , DNA/metabolismo , Fatores de Transcrição/metabolismo , Algoritmos , Sequência de Bases , Sítios de Ligação , Imunoprecipitação da Cromatina , Biologia Computacional/métodos , Aprendizado de Máquina , Modelos Estatísticos , Motivos de Nucleotídeos , Probabilidade , Técnica de Seleção de Aptâmeros , SoftwareRESUMO
Loss-of-function screening by CRISPR/Cas9 gene knockout with pooled, lentiviral guide libraries is a widely applicable method for systematic identification of genes contributing to diverse cellular phenotypes. Here, Random Sequence Labels (RSLs) are incorporated into the guide library, which act as unique molecular identifiers (UMIs) to allow massively parallel lineage tracing and lineage dropout screening. RSLs greatly improve the reproducibility of results by increasing both the precision and the accuracy of screens. They reduce the number of cells needed to reach a set statistical power, or allow a more robust screen using the same number of cells.
Assuntos
Técnicas de Inativação de Genes , Biologia de Sistemas/métodos , Sistemas CRISPR-Cas , Linhagem Celular , Biblioteca Gênica , Células HEK293 , HumanosRESUMO
The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.
Assuntos
Citosina/química , Metilação de DNA , Fosfatos de Dinucleosídeos/química , Epigênese Genética , Fatores de Transcrição/química , Ilhas de CpG , DNA/química , Genoma Humano , Humanos , Ligação Proteica , Domínios Proteicos , Técnica de Seleção de Aptâmeros , Fatores de Transcrição/classificaçãoRESUMO
To identify cell cycle regulators that enable cancer cells to replicate DNA and divide in an unrestricted manner, we performed a parallel genome-wide RNAi screen in normal and cancer cell lines. In addition to many shared regulators, we found that tumor and normal cells are differentially sensitive to loss of the histone genes transcriptional regulator CASP8AP2. In cancer cells, loss of CASP8AP2 leads to a failure to synthesize sufficient amount of histones in the S-phase of the cell cycle, resulting in slowing of individual replication forks. Despite this, DNA replication fails to arrest, and tumor cells progress in an elongated S-phase that lasts several days, finally resulting in death of most of the affected cells. In contrast, depletion of CASP8AP2 in normal cells triggers a response that arrests viable cells in S-phase. The arrest is dependent on p53, and preceded by accumulation of markers of DNA damage, indicating that nucleosome depletion is sensed in normal cells via a DNA-damage -like response that is defective in tumor cells.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Genoma , Neoplasias/metabolismo , Neoplasias/patologia , Nucleossomos/metabolismo , Interferência de RNA , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , DNA/biossíntese , Replicação do DNA , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Osteossarcoma/genética , Osteossarcoma/patologia , Fosforilação , RNA Interferente Pequeno/metabolismo , Fase S , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismoRESUMO
MOTIVATION: Transcription factor (TF) binding can be studied accurately in vivo with ChIP-exo and ChIP-Nexus experiments. Only fraction of TF binding mechanisms are yet fully understood and accurate knowledge of binding locations and patterns of TFs is key to understanding binding that is not explained by simple positional weight matrix models. ChIP-exo/Nexus experiments can also offer insight on the effect of single nucleotide polymorphism (SNP) at TF binding sites on expression of the target genes. This is an important mechanism of action for disease-causing SNPs at non-coding genomic regions. RESULTS: We describe a peak caller PeakXus that is specifically designed to leverage the increased resolution of ChIP-exo/Nexus and developed with the aim of making as few assumptions of the data as possible to allow discoveries of novel binding patterns. We apply PeakXus to ChIP-Nexus and ChIP-exo experiments performed both in Homo sapiens and in Drosophila melanogaster cell lines. We show that PeakXus consistently finds more peaks overlapping with a TF-specific recognition sequence than published methods. As an application example we demonstrate how PeakXus can be coupled with unique molecular identifiers (UMIs) to measure the effect of a SNP overlapping with a TF binding site on the in vivo binding of the TF. AVAILABILITY AND IMPLEMENTATION: Source code of PeakXus is available at https://github.com/hartonen/PeakXus CONTACT: tuomo.hartonen@helsinki.fi or jussi.taipale@ki.se.
Assuntos
Sítios de Ligação , Fatores de Transcrição , Animais , Imunoprecipitação da Cromatina , Biologia Computacional , Simulação por Computador , Drosophila melanogaster , Perfilação da Expressão Gênica , Loci Gênicos , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas , Análise de Sequência de DNARESUMO
Gene expression is regulated by transcription factors (TFs), proteins that recognize short DNA sequence motifs. Such sequences are very common in the human genome, and an important determinant of the specificity of gene expression is the cooperative binding of multiple TFs to closely located motifs. However, interactions between DNA-bound TFs have not been systematically characterized. To identify TF pairs that bind cooperatively to DNA, and to characterize their spacing and orientation preferences, we have performed consecutive affinity-purification systematic evolution of ligands by exponential enrichment (CAP-SELEX) analysis of 9,400 TF-TF-DNA interactions. This analysis revealed 315 TF-TF interactions recognizing 618 heterodimeric motifs, most of which have not been previously described. The observed cooperativity occurred promiscuously between TFs from diverse structural families. Structural analysis of the TF pairs, including a novel crystal structure of MEIS1 and DLX3 bound to their identified recognition site, revealed that the interactions between the TFs were predominantly mediated by DNA. Most TF pair sites identified involved a large overlap between individual TF recognition motifs, and resulted in recognition of composite sites that were markedly different from the individual TF's motifs. Together, our results indicate that the DNA molecule commonly plays an active role in cooperative interactions that define the gene regulatory lexicon.
Assuntos
DNA/genética , DNA/metabolismo , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Cristalografia por Raios X , Regulação da Expressão Gênica/genética , Humanos , Dados de Sequência Molecular , Motivos de Nucleotídeos/genética , Reprodutibilidade dos Testes , Especificidade por Substrato/genéticaRESUMO
Cohesin is present in almost all active enhancer regions, where it is associated with transcription factors. Cohesin frequently colocalizes with CTCF (CCCTC-binding factor), affecting genomic stability, expression and epigenetic homeostasis. Cohesin subunits are mutated in cancer, but CTCF/cohesin-binding sites (CBSs) in DNA have not been examined for mutations. Here we report frequent mutations at CBSs in cancers displaying a mutational signature where mutations in Aâ¢T base pairs predominate. Integration of whole-genome sequencing data from 213 colorectal cancer (CRC) samples and chromatin immunoprecipitation sequencing (ChIP-exo) data identified frequent point mutations at CBSs. In contrast, CRCs showing an ultramutator phenotype caused by defects in the exonuclease domain of DNA polymerase É (POLE) displayed significantly fewer mutations at and adjacent to CBSs. Analysis of public data showed that multiple cancer types accumulate CBS mutations. CBSs are a major mutational hotspot in the noncoding cancer genome.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Proteínas Repressoras/fisiologia , Sítios de Ligação , Fator de Ligação a CCCTC , Neoplasias Colorretais , Sequência Consenso , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Mutação Puntual , Sequências Reguladoras de Ácido Nucleico , CoesinasRESUMO
Divergent morphology of species has largely been ascribed to genetic differences in the tissue-specific expression of proteins, which could be achieved by divergence in cis-regulatory elements or by altering the binding specificity of transcription factors (TFs). The relative importance of the latter has been difficult to assess, as previous systematic analyses of TF binding specificity have been performed using different methods in different species. To address this, we determined the binding specificities of 242 Drosophila TFs, and compared them to human and mouse data. This analysis revealed that TF binding specificities are highly conserved between Drosophila and mammals, and that for orthologous TFs, the similarity extends even to the level of very subtle dinucleotide binding preferences. The few human TFs with divergent specificities function in cell types not found in fruit flies, suggesting that evolution of TF specificities contributes to emergence of novel types of differentiated cells.
Assuntos
Evolução Biológica , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Drosophila , Duplicação Gênica , Humanos , Camundongos , Filogenia , Técnica de Seleção de Aptâmeros , Homologia de Sequência de AminoácidosRESUMO
Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at very high frequency (â¼70%) in uterine leiomyomas. However, the influence of these mutations on Mediator function and the molecular basis for their tumorigenic potential remain unknown. To clarify the impact of these mutations, we used affinity-purification mass spectrometry to establish the global protein-protein interaction profiles for both wild-type and mutant MED12. We found that uterine leiomyoma-linked mutations in MED12 led to a highly specific decrease in its association with Cyclin C-CDK8/CDK19 and loss of Mediator-associated CDK activity. Mechanistically, this occurs through disruption of a MED12-Cyclin C binding interface that we also show is required for MED12-mediated stimulation of Cyclin C-dependent CDK8 kinase activity. These findings indicate that uterine leiomyoma-linked mutations in MED12 uncouple Cyclin C-CDK8/19 from core Mediator and further identify the MED12/Cyclin C interface as a prospective therapeutic target in CDK8-driven cancers.
Assuntos
Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Leiomioma/genética , Complexo Mediador/genética , Complexo Mediador/metabolismo , Neoplasias Uterinas/genética , Ciclina C/metabolismo , Feminino , Células HEK293 , Humanos , Leiomioma/metabolismo , Leiomioma/patologia , Mutagênese Sítio-Dirigida , Ligação Proteica , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologiaRESUMO
During cell division, transcription factors (TFs) are removed from chromatin twice, during DNA synthesis and during condensation of chromosomes. How TFs can efficiently find their sites following these stages has been unclear. Here, we have analyzed the binding pattern of expressed TFs in human colorectal cancer cells. We find that binding of TFs is highly clustered and that the clusters are enriched in binding motifs for several major TF classes. Strikingly, almost all clusters are formed around cohesin, and loss of cohesin decreases both DNA accessibility and binding of TFs to clusters. We show that cohesin remains bound in S phase, holding the nascent sister chromatids together at the TF cluster sites. Furthermore, cohesin remains bound to the cluster sites when TFs are evicted in early M phase. These results suggest that cohesin-binding functions as a cellular memory that promotes re-establishment of TF clusters after DNA replication and chromatin condensation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Motivos de Nucleotídeos , CoesinasRESUMO
In this work, we map the transcriptional targets of 107 previously identified Drosophila genes whose loss caused the strongest cell-cycle phenotypes in a genome-wide RNA interference screen and mine the resulting data computationally. Besides confirming existing knowledge, the analysis revealed several regulatory systems, among which were two highly-specific and interconnected feedback circuits, one between the ribosome and the proteasome that controls overall protein homeostasis, and the other between the ribosome and Myc/Max that regulates the protein synthesis capacity of cells. We also identified a set of genes that alter the timing of mitosis without affecting gene expression, indicating that the cyclic transcriptional program that produces the components required for cell division can be partially uncoupled from the cell division process itself. These genes all have a function in a pathway that regulates the phosphorylation state of Cdk1. We provide evidence showing that this pathway is involved in regulation of cell size, indicating that a Cdk1-regulated cell size checkpoint exists in metazoans.
Assuntos
Ciclo Celular/fisiologia , Tamanho Celular , Drosophila/genética , Redes Reguladoras de Genes/genética , Animais , Proteína Quinase CDC2/metabolismo , Ciclo Celular/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Análise por Conglomerados , Drosophila/fisiologia , Retroalimentação Fisiológica/fisiologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Análise em Microsséries , Mitose/genética , Oligonucleotídeos/genética , Fosforilação , Interferência de RNA , Análise de Sequência de RNARESUMO
Although the proteins that read the gene regulatory code, transcription factors (TFs), have been largely identified, it is not well known which sequences TFs can recognize. We have analyzed the sequence-specific binding of human TFs using high-throughput SELEX and ChIP sequencing. A total of 830 binding profiles were obtained, describing 239 distinctly different binding specificities. The models represent the majority of human TFs, approximately doubling the coverage compared to existing systematic studies. Our results reveal additional specificity determinants for a large number of factors for which a partial specificity was known, including a commonly observed A- or T-rich stretch that flanks the core motifs. Global analysis of the data revealed that homodimer orientation and spacing preferences, and base-stacking interactions, have a larger role in TF-DNA binding than previously appreciated. We further describe a binding model incorporating these features that is required to understand binding of TFs to DNA.
Assuntos
Imunoprecipitação da Cromatina , Modelos Biológicos , Técnica de Seleção de Aptâmeros , Fatores de Transcrição/metabolismo , Animais , DNA/química , Humanos , Cadeias de Markov , Camundongos , Filogenia , Fatores de Transcrição/genéticaRESUMO
Fungal infection of barley and malt, particularly by the Fusarium species, is a direct cause of spontaneous overfoaming of beer, referred to as gushing. We have shown previously that small fungal proteins, hydrophobins, act as gushing-inducing factors in beer. The aim of our present study was to isolate and characterize hydrophobins from a gushing-active fungus, Fusarium graminearum (teleomorph Gibberella zeae) and related species. We generated profile hidden Markov models (profile HMMs) for the hydrophobin classes Ia, Ib and II from the multiple sequence alignments of their known members available in public domain databases. We searched the published Fusarium graminearum genome with the Markov models. The best matching sequences and the corresponding genes were isolated from F. graminearum and the related species F. culmorum and F. poae by PCR and characterized. One each of the putative F. graminearum and F. poae hydrophobin genes were expressed in the heterologous host Trichoderma reesei. The proteins corresponding to the genes were purified and identified as hydrophobins and named GzHYD5 and FpHYD5, respectively. Concentrations of 0.003 ppm of these hydrophobins were observed to induce vigorous beer gushing.
Assuntos
Cerveja/microbiologia , Proteínas Fúngicas/metabolismo , Fusarium/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA Fúngico/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Fusarium/metabolismo , Genes Fúngicos , Cadeias de Markov , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Counting individual RNA or DNA molecules is difficult because they are hard to copy quantitatively for detection. To overcome this limitation, we applied unique molecular identifiers (UMIs), which make each molecule in a population distinct, to genome-scale human karyotyping and mRNA sequencing in Drosophila melanogaster. Use of this method can improve accuracy of almost any next-generation sequencing method, including chromatin immunoprecipitation-sequencing, genome assembly, diagnostics and manufacturing-process control and monitoring.