RESUMEN
Epigenetic silenced alleles of the Arabidopsis SUPERMAN locus (the clark kent alleles) are associated with dense hypermethylation at noncanonical cytosines (CpXpG and asymmetric sites, where X = A, T, C, or G). A genetic screen for suppressors of a hypermethylated clark kent mutant identified nine loss-of-function alleles of CHROMOMETHYLASE3 (CMT3), a novel cytosine methyltransferase homolog. These cmt3 mutants display a wild-type morphology but exhibit decreased CpXpG methylation of the SUP gene and of other sequences throughout the genome. They also show reactivated expression of endogenous retrotransposon sequences. These results show that a non-CpG DNA methyltransferase is responsible for maintaining epigenetic gene silencing.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Metilación de ADN , ADN-Citosina Metilasas/genética , ADN-Citosina Metilasas/metabolismo , Silenciador del Gen , Oligonucleótidos/metabolismo , Factores de Transcripción/genética , Alelos , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Islas de CpG , Cruzamientos Genéticos , Citosina/metabolismo , ADN-Citosina Metilasas/química , Fosfatos de Dinucleósidos/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Datos de Secuencia Molecular , Mutagénesis , Fenotipo , Estructura Terciaria de Proteína , RetroelementosRESUMEN
The most highly conserved regions of proteins can be represented as blocks of aligned sequence segments, typically with multiple blocks for a given protein family. The Blocks Database World Wide Web (http://blocks.fhcrc.org) and e-mail (blocks@blocks. fhcrc.org) servers provide tools to search DNA and protein queries against the Blocks+ Database of multiple alignments. We describe features for detection of distant relationships using blocks. Blocks+ includes protein families from the PROSITE, Prints, Pfam-A, ProDom and Domo databases. Other features include searching Blocks+ with the BLIMPS and NCBI's IMPALA programs, sequence logos, phylogenetic trees, three-dimensional display of blocks on PDB structures, and a polymerase chain reaction (PCR) primer design strategy based on blocks.
Asunto(s)
Bases de Datos Factuales , Proteínas/análisis , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ProteínaRESUMEN
With the accumulation of large-scale sequence data, emphasis in genomics has shifted from determining gene structure to testing gene function, and this relies on reverse genetic methodology. Here we explore the feasibility of screening for chemically induced mutations in target sequences in Arabidopsis thaliana. Our TILLING (Targeting Induced Local Lesions IN Genomes) method combines the efficiency of ethyl methanesulfonate (EMS)-induced mutagenesis with the ability of denaturing high-performance liquid chromatography (DHPLC) to detect base pair changes by heteroduplex analysis. Importantly, this method generates a wide range of mutant alleles, is fast and automatable, and is applicable to any organism that can be chemically mutagenized.
Asunto(s)
Arabidopsis/genética , ADN (Citosina-5-)-Metiltransferasas , Mutagénesis , Alelos , Sustitución de Aminoácidos , Arabidopsis/efectos de los fármacos , Disparidad de Par Base , Emparejamiento Base , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Clonación Molecular , Codón de Terminación , Secuencia Conservada , Metilasas de Modificación del ADN/genética , Cartilla de ADN , Metanosulfonato de Etilo/farmacología , Ingeniería Genética/métodos , Intrones , Mutación Puntual , Reacción en Cadena de la PolimerasaRESUMEN
The trithorax group gene brahma (brm) encodes an activator of Drosophila homeotic genes that functions as the ATPase subunit of a large protein complex. To determine if BRM physically interacts with other trithorax group proteins, we purified the BRM complex from Drosophila embryos and analyzed its subunit composition. The BRM complex contains at least seven major polypeptides. Surprisingly, the majority of the subunits of the BRM complex are not encoded by trithorax group genes. Furthermore, a screen for enhancers of a dominant-negative brm mutation identified only one trithorax group gene, moira (mor), that appears to be essential for brm function in vivo. Four of the subunits of the BRM complex are related to subunits of the yeast chromatin remodeling complexes SWI/SNF and RSC. The BRM complex is even more highly related to the human BRG1 and hBRM complexes, but lacks the subunit heterogeneity characteristic of these complexes. We present biochemical evidence for the existence of two additional complexes containing trithorax group proteins: a 2 MDa ASH1 complex and a 500 kDa ASH2 complex. These findings suggest that BRM plays a role in chromatin remodeling that is distinct from the function of most other trithorax group proteins.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Drosophila , Drosophila/metabolismo , Proteínas de Insectos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Southern Blotting , Western Blotting , Cruzamientos Genéticos , ADN Helicasas , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Drosophila/embriología , Drosophila/genética , Etiquetas de Secuencia Expresada , Proteínas del Grupo de Alta Movilidad , N-Metiltransferasa de Histona-Lisina , Humanos , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas Nucleares/química , Pruebas de Precipitina , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/química , Levaduras/genéticaRESUMEN
We describe a new primer design strategy for PCR amplification of unknown targets that are related to multiply-aligned protein sequences. Each primer consists of a short 3' degenerate core region and a longer 5' consensus clamp region. Only 3-4 highly conserved amino acid residues are necessary for design of the core, which is stabilized by the clamp during annealing to template molecules. During later rounds of amplification, the non-degenerate clamp permits stable annealing to product molecules. We demonstrate the practical utility of this hybrid primer method by detection of diverse reverse transcriptase-like genes in a human genome, and by detection of C5DNA methyltransferase homologs in various plant DNAs. In each case, amplified products were sufficiently pure to be cloned without gel fractionation. This COnsensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) strategy has been implemented as a computer program that is accessible over the World Wide Web (http://blocks.fhcrc.org/codehop.html) and is directly linked from the BlockMaker multiple sequence alignment site for hybrid primer prediction beginning with a set of related protein sequences.
Asunto(s)
Metilasas de Modificación del ADN/química , Cartilla de ADN , Evolución Molecular , Filogenia , ADN Polimerasa Dirigida por ARN/química , Secuencia de Aminoácidos , Animales , Artritis Reumatoide/genética , Secuencia de Bases , Codón , Redes de Comunicación de Computadores , Secuencia de Consenso , Secuencia Conservada , Metilasas de Modificación del ADN/genética , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , ADN Polimerasa Dirigida por ARN/genética , Sarcoma de Kaposi/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Programas InformáticosRESUMEN
During most of Drosophila development the regulation of homeotic gene transcription is controlled by two groups of regulatory genes, the trithorax group of activators and the Polycomb group of repressors. brahma (brm), a member of the trithorax group, encodes a protein related to the yeast SWI2/SNF2 protein, a subunit of a protein complex that assists sequence-specific activator proteins by alleviating the repressive effects of chromatin. To learn more about the molecular mechanisms underlying the regulation of homeotic gene transcription, we have investigated whether a similar complex exists in flies. We identified the Drosophila snr1 gene, a potential homologue of the yeast SNF5 gene that encodes a subunit of the yeast SWI/SNF complex. The snr1 gene is essential and genetically interacts with brm and trithorax (trx), suggesting cooperation in regulating homeotic gene transcription. The spatial and temporal patterns of expression of snr1 are similar to those of brm. The snr1 and brm proteins are present in a large (> 2 x 10(6) Da) complex, and they co-immunoprecipitate from Drosophila extracts. These findings provide direct evidence for conservation of the SWI/SNF complex in higher eucaryotes and suggest that the Drosophila brm/snr1 complex plays an important role in maintaining homeotic gene transcription during development by counteracting the repressive effects of chromatin.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares , Transactivadores/análisis , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Cromosómicas no Histona , Clonación Molecular , Femenino , Genes de Insecto/genética , Masculino , Datos de Secuencia Molecular , Peso Molecular , ARN Mensajero/análisis , Proteína SMARCB1 , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Levaduras/genéticaRESUMEN
The Drosophila brahma (brm) gene encodes an activator of homeotic genes that is highly related to the yeast transcriptional activator SWI2 (SNF2), a potential helicase. To determine whether brm is a functional homolog of SWI2 or merely a member of a family of SWI2-related genes, we searched for additional Drosophila genes related to SWI2 and examined their function in yeast cells. In addition to brm, we identified one other Drosophila relative of SWI2: the closely related ISWI gene. The 1,027-residue ISWI protein contains the DNA-dependent ATPase domain characteristic of the SWI2 protein family but lacks the three other domains common to brm and SWI2. In contrast, the ISWI protein is highly related (70% identical) to the human hSNF2L protein over its entire length, suggesting that they may be functional homologs. The DNA-dependent ATPase domains of brm and SWI2, but not ISWI, are functionally interchangeable; a chimeric SWI2-brm protein partially rescued the slow growth of swi2- cells and supported transcriptional activation mediated by the glucocorticoid receptor in vivo in yeast cells. These findings indicate that brm is the closest Drosophila relative of SWI2 and suggest that brm and SWI2 play similar roles in transcriptional activation.