RESUMEN
Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.
Asunto(s)
Cromosomas Artificiales de Levadura/fisiología , Genoma Fúngico , Saccharomyces cerevisiae/genética , Segregación Cromosómica , Cromosomas Artificiales de Levadura/química , Cromosomas Artificiales de Levadura/genética , Medios de Cultivo/química , Replicación del ADN , Glicerol , Proteómica , Saccharomyces cerevisiae/crecimiento & desarrollo , Análisis de Secuencia de ADN , Biología Sintética , TranscriptomaRESUMEN
DNA topoisomerases are thought to play a critical role in transcription, replication and recombination as well as in the condensation and segregation of sister duplexes during cell division. Here, we used high-resolution two-dimensional agarose gel electrophoresis to study the replication intermediates and final products of small circular and linear minichromosomes of Saccharomyces cerevisiae in the presence and absence of DNA topoisomerase 2. The results obtained confirmed that whereas for circular minichromosomes, catenated sister duplexes accumulated in the absence of topoisomerase 2, linear YACs were able to replicate and segregate regardless of this topoisomerase. The patterns of replication intermediates for circular and linear YACs displayed significant differences suggesting that DNA supercoiling might play a key role in the modulation of replication fork progression. Altogether, this data supports the notion that for linear chromosomes the torsional tension generated by transcription and replication dissipates freely throughout the telomeres.
Asunto(s)
Segregación Cromosómica/genética , Segregación Cromosómica/fisiología , Cromosomas Artificiales de Levadura/genética , Replicación del ADN/genética , Replicación del ADN/fisiología , ADN-Topoisomerasas de Tipo II/metabolismo , Cromosomas Artificiales de Levadura/fisiología , ADN Circular/genética , ADN Circular/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Telómero/metabolismoRESUMEN
We generated a line of transgenic mice using a yeast artificial chromosome containing the Ret mutation responsible for the multiple endocrine neoplasia type 2B syndrome (MEN 2B). The resulting animals did not develop any of the expected neoplasms associated with MEN 2B. Transgenic animals were then bred with animals lacking murine Ret (Ret(M)) to further evaluate the function of human mutated Ret (Ret(H)(2B)) in the murine context. Whereas mice lacking Ret(M) exhibit intestinal aganglionosis and the absence of kidneys with other genitourinary anomalies, expression of the Ret(H)(2B) transgene in Ret(M)-deficient mice allowed significant renal development with a partial rescue of the enteric nervous system. These Ret(H)(2B)-positive/Ret(M)-deficient mice exhibit normal Ret expression and survive longer than Ret(M)-deficient mice, but still die at 3 to 5 days of age with evidence of enterocolitis. We conclude that the normal expression of a human Ret proto-oncogene with the MEN 2B mutation does not cause any features of MEN 2B in mice. Although the gene is normally expressed in the appropriate target tissues, there is incomplete phenotypic rescue in mice lacking murine Ret. These results suggest important interspecies differences between humans and mice in the function of the Ret oncogene.
Asunto(s)
Cromosomas Artificiales de Levadura/fisiología , Neoplasia Endocrina Múltiple Tipo 2b/genética , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/deficiencia , Proteínas Tirosina Quinasas Receptoras/genética , Animales , Secuencia de Bases , Cartilla de ADN , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Neoplasia Endocrina Múltiple Tipo 2b/prevención & control , Reacción en Cadena de la Polimerasa , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-ret , Mapeo RestrictivoRESUMEN
Checkpoint controls coordinate entry into mitosis with the completion of DNA replication. Depletion of nucleotide precursors by treatment with the drug hydroxyurea triggers such a checkpoint response. However, it is not clear whether the signal for this hydroxyurea-induced checkpoint pathway is the presence of unreplicated DNA, or rather the persistence of single-stranded or damaged DNA. In a yeast artificial chromosome (YAC) we have engineered an approximately 170 kb region lacking efficient replication origins that allows us to explore the specific effects of unreplicated DNA on cell cycle progression. Replication of this YAC extends the length of S phase and causes cells to engage an S/M checkpoint. In the absence of Rad9 the YAC becomes unstable, undergoing deletions within the origin-free region.
Asunto(s)
Cromosomas Artificiales de Levadura/fisiología , Genes cdc/fisiología , Mitosis/genética , Proteínas Serina-Treonina Quinasas , Origen de Réplica/genética , Fase S/genética , Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2 , Daño del ADN/genética , Replicación del ADN/genética , Eliminación de Gen , Genes Fúngicos/fisiología , Hidroxiurea , Inhibidores de la Síntesis del Ácido Nucleico , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Saccharomyces cerevisiaeRESUMEN
Yeast artificial chromosomes (YACs) are a common tool for cloning eukaryotic DNA. The manner by which large pieces of foreign DNA are assimilated by yeast cells into a functional chromosome is poorly understood, as is the reason why some of them are stably maintained and some are not. We examined the replication of a stable YAC containing a 240-kb insert of DNA from the human T-cell receptor beta locus. The human insert contains multiple sites that serve as origins of replication. The activity of these origins appears to require the yeast ARS consensus sequence and, as with yeast origins, additional flanking sequences. In addition, the origins in the human insert exhibit a spacing, a range of activation efficiencies, and a variation in times of activation during S phase similar to those found for normal yeast chromosomes. We propose that an appropriate combination of replication origin density, activation times, and initiation efficiencies is necessary for the successful maintenance of YAC inserts.
Asunto(s)
Cromosomas Artificiales de Levadura/fisiología , Replicación del ADN/fisiología , Origen de Réplica , Cromosomas Humanos Par 7 , Mapeo Contig , Electroforesis en Gel de Agar , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T/genética , Humanos , Cinética , Modelos Genéticos , Mutagénesis Sitio-Dirigida , Factores de TiempoRESUMEN
Several isogenic strains with defects in recombination/repair genes (RAD1, RAD50, RAD51, RAD52, RAD54, and RAD55) were examined for their ability to propagate accurately a variety of linear and circular yeast artificial chromosomes (YACs) containing human DNA inserts. To assess YAC stability, the human DNA inserts were internally marked by an ADE2-pBR-URA3 cassette. Following selection for Ura- clones on 5-fluoroorotic acid containing medium, the following types of YAC deletions were identified: (i) those caused by homologous recombination with a telomeric pBR sequence; (ii) internal deletions, presumed to occur by recombination between commonly occurring DNA repeats such as Alu and LINE sequences; and (iii) deletions leading to loss of part of a YAC arm. rad52 host strains, but not other recombination-deficient strains, decreased the rate of all types of YAC deletions 25- to 400-fold. We have also developed and tested kar1 strains with a conditional RAD52 gene that allow transfer of a YAC from any host into a recombination-deficient background. These strains provide an efficient tool for stabilization of YACs and are useful for allowing additional recombinational modification of YACs.
Asunto(s)
Cromosomas Artificiales de Levadura/metabolismo , Cromosomas Artificiales de Levadura/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Saccharomyces cerevisiae , Mapeo Cromosómico , ADN Helicasas , Cartilla de ADN , Enzimas Reparadoras del ADN , Proteínas de Unión al ADN/fisiología , Endonucleasas/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Galactosa/metabolismo , Eliminación de Gen , Genotipo , Glucosa/metabolismo , Humanos , Mitosis/genética , Modelos Biológicos , Mutagénesis Insercional , Recombinasa Rad51 , Proteína Recombinante y Reparadora de ADN Rad52 , Transformación Genética , Levaduras/genéticaRESUMEN
Plasmids carrying the Epstein-Barr virus origin of plasmid replication (oriP) have been shown to replicate autonomously in latently infected human cells (J. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. USA 81:3806-3810, 1984). We demonstrate that addition of this domain is sufficient for stable episomal maintenance of yeast artificial chromosomes (YACs), up to at least 660 kb, in human cells expressing the viral protein EBNA-1. To better approximate the latent viral genome, YACs were circularized before addition of the oriP domain by homologous recombination in yeast cells. The resulting OriPYACs were maintained as extrachromosomal molecules over long periods in selection; a 90-kb OriPYAC was unrearranged in all cell lines analyzed, whereas the intact form of a 660-kb molecule was present in two of three cell lines. The molecules were also relatively stable in the absence of selection. This finding indicates that the oriP-EBNA-1 interaction is sufficient to stabilize episomal molecules of at least 660 kb and that such elements do not undergo rearrangements over time. Fluorescence in situ hybridization analysis demonstrated a close association of OriPYACs, some of which were visible as pairs, with host cell chromosomes, suggesting that the episomes replicate once per cell cycle and that stability is achieved by attachment to host chromosomes, as suggested for the viral genome. The wide availability of YAC libraries, the ease of manipulation of cloned sequences in yeast cells, and the episomal stability make OriPYACs ideal for studying gene function and control of gene expression.
Asunto(s)
Cromosomas Artificiales de Levadura/fisiología , Herencia Extracromosómica , Plásmidos/fisiología , Antígenos Virales/biosíntesis , Antígenos Virales/genética , Línea Celular Transformada , Cromosomas Humanos/ultraestructura , ADN Circular/genética , ADN de Hongos/genética , ADN Recombinante/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Antígenos Nucleares del Virus de Epstein-Barr , Herpesvirus Humano 4/genética , Humanos , Hibridación Fluorescente in Situ , Riñón/citología , Origen de RéplicaRESUMEN
A chromosome bisection method was constructed using yeast artificial chromosomes (YAC) and a GAL1-promoted site-specific recombination system. This method was applied to bisect chromosome IV into the left and right parts of the centromere region. The bisection occurred at frequencies of about 10% when the recombination site DNAs were integrated onto YAC and chromosome IV in the same direction, but were less than 10(-3) when they were in opposite directions. Reconstruction of the original chromosome IV from the bisected chromosomes was also induced by galactose at high frequencies. Loss of the left part chromosome was found at the frequencies of 0.9 x 10(-3) after hybrid cells between the chromosome-bisected strain and a normal haploid were subcultivated in a complete medium. The bisection and reconstruction of chromosome IV and deletion of the left part chromosome were demonstrated by electrophoretical karyotyping.