RESUMEN
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.
Asunto(s)
Edición Génica , Factores de Transcripción , Sitios de Unión/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Regiones Promotoras Genéticas/genética , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Proteínas Proto-Oncogénicas c-myc , Ribosomas , Animales , Proliferación Celular/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ribosomas/metabolismo , Activación Transcripcional , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción , Sitios de Unión/genética , Genoma Humano/genética , Humanos , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , ARN/química , ARN/metabolismo , Secuencia de Bases , Genoma Humano , Humanos , Conformación de Ácido Nucleico , Motivos de Nucleótidos , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Ribonucleasas/química , Ribonucleasas/metabolismo , Técnica SELEX de Producción de AptámerosRESUMEN
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.
Asunto(s)
Desequilibrio Alélico , Neoplasias Colorrectales/genética , Predisposición Genética a la Enfermedad , Sistemas CRISPR-Cas , Aberraciones Cromosómicas , Cromosomas Humanos Par 8 , Neoplasias Colorrectales/patología , Variaciones en el Número de Copia de ADN , Dinamarca , Perfilación de la Expresión Génica , Genómica , Genotipo , Humanos , Pérdida de Heterocigocidad , Repeticiones de Microsatélite , Fenotipo , Mutación Puntual , Proteínas Proto-Oncogénicas p21(ras)/genética , ARN Interferente Pequeño/genética , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Secuenciación Completa del GenomaRESUMEN
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.
Asunto(s)
Cromatina/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Biotecnología , Diferenciación Celular , Cromatina/genética , ADN/genética , ADN/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Especificidad de la Especie , Distribución TisularRESUMEN
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.
Asunto(s)
ADN/química , ADN/metabolismo , Factores de Transcripción/metabolismo , Algoritmos , Secuencia de Bases , Sitios de Unión , Inmunoprecipitación de Cromatina , Biología Computacional/métodos , Aprendizaje Automático , Modelos Estadísticos , Motivos de Nucleótidos , Probabilidad , Técnica SELEX de Producción de Aptámeros , Programas InformáticosRESUMEN
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.
Asunto(s)
Técnicas de Inactivación de Genes , Biología de Sistemas/métodos , Sistemas CRISPR-Cas , Línea Celular , Biblioteca de Genes , Células HEK293 , HumanosRESUMEN
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.
Asunto(s)
Citosina/química , Metilación de ADN , Fosfatos de Dinucleósidos/química , Epigénesis Genética , Factores de Transcripción/química , Islas de CpG , ADN/química , Genoma Humano , Humanos , Unión Proteica , Dominios Proteicos , Técnica SELEX de Producción de Aptámeros , Factores de Transcripción/clasificaciónRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Genoma , Neoplasias/metabolismo , Neoplasias/patología , Nucleosomas/metabolismo , Interferencia de ARN , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , ADN/biosíntesis , Replicación del ADN , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Osteosarcoma/genética , Osteosarcoma/patología , Fosforilación , ARN Interferente Pequeño/metabolismo , Fase S , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
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.
Asunto(s)
Sitios de Unión , Factores de Transcripción , Animales , Inmunoprecipitación de Cromatina , Biología Computacional , Simulación por Computador , Drosophila melanogaster , Perfilación de la Expresión Génica , Sitios Genéticos , Humanos , Unión Proteica , Mapeo de Interacción de Proteínas , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
ADN/genética , ADN/metabolismo , Especificidad por Sustrato , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Cristalografía por Rayos X , Regulación de la Expresión Génica/genética , Humanos , Datos de Secuencia Molecular , Motivos de Nucleótidos/genética , Reproducibilidad de los Resultados , Especificidad por Sustrato/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/fisiología , Proteínas Represoras/fisiología , Sitios de Unión , Factor de Unión a CCCTC , Neoplasias Colorrectales , Secuencia de Consenso , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Mutación Puntual , Secuencias Reguladoras de Ácidos Nucleicos , CohesinasRESUMEN
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.
Asunto(s)
Evolución Biológica , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Drosophila , Duplicación de Gen , Humanos , Ratones , Filogenia , Técnica SELEX de Producción de Aptámeros , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Leiomioma/genética , Complejo Mediador/genética , Complejo Mediador/metabolismo , Neoplasias Uterinas/genética , Ciclina C/metabolismo , Femenino , Células HEK293 , Humanos , Leiomioma/metabolismo , Leiomioma/patología , Mutagénesis Sitio-Dirigida , Unión Proteica , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologíaRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Proteínas Cromosómicas no Histona/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Motivos de Nucleótidos , CohesinasRESUMEN
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.
Asunto(s)
Ciclo Celular/fisiología , Tamaño de la Célula , Drosophila/genética , Redes Reguladoras de Genes/genética , Animales , Proteína Quinasa CDC2/metabolismo , Ciclo Celular/genética , Línea Celular , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Drosophila/fisiología , Retroalimentación Fisiológica/fisiología , Citometría de Flujo , Perfilación de la Expresión Génica , Análisis por Micromatrices , Mitosis/genética , Oligonucleótidos/genética , Fosforilación , Interferencia de ARN , Análisis de Secuencia de ARNRESUMEN
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.
Asunto(s)
Inmunoprecipitación de Cromatina , Modelos Biológicos , Técnica SELEX de Producción de Aptámeros , Factores de Transcripción/metabolismo , Animales , ADN/química , Humanos , Cadenas de Markov , Ratones , Filogenia , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Cerveza/microbiología , Proteínas Fúngicas/metabolismo , Fusarium/genética , Secuencia de Aminoácidos , Clonación Molecular , ADN de Hongos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Fusarium/metabolismo , Genes Fúngicos , Cadenas de Markov , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
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.