RESUMEN
Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the â¼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.
Asunto(s)
Cromosomas Artificiales de Levadura , Genoma Fúngico , Saccharomyces cerevisiae , Perfilación de la Expresión Génica , Proteómica , Saccharomyces cerevisiae/genética , Biología Sintética , ARN de Transferencia/genética , Cromosomas Artificiales de Levadura/genéticaRESUMEN
The fission yeast species Schizosaccharomyces japonicus is currently divided into two varieties-S. japonicus var. japonicus and S. japonicus var. versatilis. Here we examine the var. versatilis isolate CBS5679. The CBS5679 genome shows 88% identity to the reference genome of S. japonicus var. japonicus at the coding sequence level, with phylogenetic analyses suggesting that it has split from the S. japonicus lineage 25 million years ago. The CBS5679 genome contains a reciprocal translocation between chromosomes 1 and 2, together with several large inversions. The products of genes linked to the major translocation are associated with 'metabolism' and 'cellular assembly' ontology terms. We further show that CBS5679 does not generate viable progeny with the reference strain of S. japonicus. Although CBS5679 shares closer similarity to the 'type' strain of var. versatilis as compared to S. japonicus, it is not identical to the type strain, suggesting population structure within var. versatilis. We recommend that the taxonomic status of S. japonicus var. versatilis is raised, with it being treated as a separate species, Schizosaccharomyces versatilis.
Asunto(s)
Schizosaccharomyces , Schizosaccharomyces/genética , Filogenia , Evolución BiológicaRESUMEN
Schizosaccharomyces japonicus belongs to the single-genus class Schizosaccharomycetes, otherwise known as "fission yeasts." As part of a composite model system with its widely studied S. pombe sister species, S. japonicus has provided critical insights into the workings and the evolution of cell biological mechanisms. Furthermore, its divergent biology makes S. japonicus a valuable model organism in its own right. However, the currently available genome assembly contains gaps and has been unable to resolve centromeres and other repeat-rich chromosomal regions. Here we present a telomere-to-telomere long-read genome assembly of the S. japonicus genome. This includes the three megabase-length chromosomes, with centromeres hundreds of kilobases long, rich in 5S ribosomal RNA genes, transfer RNA genes, long terminal repeats, and short repeats. We identify a gene-sparse region on chromosome 2 that resembles a 331 kb centromeric duplication. We revise the genome size of S. japonicus to at least 16.6 Mb and possibly up to 18.12 Mb, at least 30% larger than previous estimates. Our whole genome assembly will support the growing S. japonicus research community and facilitate research in new directions, including centromere and DNA repeat evolution, and yeast comparative genomics.
Asunto(s)
Schizosaccharomyces , Schizosaccharomyces/genética , Telómero/genética , Centrómero/genéticaRESUMEN
A eukaryotic chromosome relies on the function of multiple spatially distributed DNA replication origins for its stable inheritance. The spatial location of an origin is determined by the chromosomal position of an MCM complex, the inactive form of the DNA replicative helicase that is assembled onto DNA in G1-phase (also known as origin licensing). While the biochemistry of origin licensing is understood, the mechanisms that promote an adequate spatial distribution of MCM complexes across chromosomes are not. We have elucidated a role for the Sir2 histone deacetylase in establishing the normal distribution of MCM complexes across Saccharomyces cerevisiae chromosomes. In the absence of Sir2, MCM complexes accumulated within both early-replicating euchromatin and telomeric heterochromatin, and replication activity within these regions was enhanced. Concomitantly, the duplication of several regions of late-replicating euchromatin were delayed. Thus, Sir2-mediated attenuation of origin licensing within both euchromatin and telomeric heterochromatin established the normal spatial distribution of origins across yeast chromosomes important for normal genome duplication.
Asunto(s)
Eucromatina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona , Cromosomas , ADN Helicasas , Replicación del ADN , Heterocromatina , Origen de Réplica/genéticaRESUMEN
Saccharomyces cerevisiae exhibits gene expression homeostasis, which is defined as the buffering of transcription levels against changes in DNA copy number during the S phase of the cell cycle. It has been suggested that S. cerevisiae employs an active mechanism to maintain gene expression homeostasis through Rtt109-Asf1-dependent acetylation of histone H3 on lysine 56 (H3K56). Here, we show that gene expression homeostasis can be achieved independently of H3K56 acetylation by Tos4 (Target of Swi6-4). Using Nanostring technology, we establish that Tos4-dependent gene expression homeostasis depends on its forkhead-associated (FHA) domain, which is a phosphopeptide recognition domain required to bind histone deacetylases (HDACs). We demonstrate that the mechanism of Tos4-dependent gene expression homeostasis requires its interaction with the Rpd3L HDAC complex. However, this is independent of Rpd3's well-established roles in both histone deacetylation and controlling the DNA replication timing program, as established by deep sequencing of Fluorescence-Activated Cell Sorted (FACS) S and G2 phase populations. Overall, our data reveals that Tos4 mediates gene expression homeostasis through its FHA domain-dependent interaction with the Rpd3L complex, which is independent of H3K56ac.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Homeostasis , Lisina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilación , Histona Acetiltransferasas/genética , Histonas/genética , Lisina/genética , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Replication of eukaryotic genomes is highly stochastic, making it difficult to determine the replication dynamics of individual molecules with existing methods. We report a sequencing method for the measurement of replication fork movement on single molecules by detecting nucleotide analog signal currents on extremely long nanopore traces (D-NAscent). Using this method, we detect 5-bromodeoxyuridine (BrdU) incorporated by Saccharomyces cerevisiae to reveal, at a genomic scale and on single molecules, the DNA sequences replicated during a pulse-labeling period. Under conditions of limiting BrdU concentration, D-NAscent detects the differences in BrdU incorporation frequency across individual molecules to reveal the location of active replication origins, fork direction, termination sites, and fork pausing/stalling events. We used sequencing reads of 20-160 kilobases to generate a whole-genome single-molecule map of DNA replication dynamics and discover a class of low-frequency stochastic origins in budding yeast. The D-NAscent software is available at https://github.com/MBoemo/DNAscent.git .
Asunto(s)
Replicación del ADN , Genoma Fúngico , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Nanoporos , Saccharomyces cerevisiae/genética , Bromodesoxiuridina/metabolismo , ADN de Hongos/genética , Genoma , Programas InformáticosRESUMEN
Biological systems are made up of components that change their actions (and interactions) over time and coordinate with other components nearby. Together with a large state space, the complexity of this behaviour can make it difficult to create concise mathematical models that can be easily extended or modified. This paper introduces the Beacon Calculus, a process algebra designed to simplify the task of modelling interacting biological components. Its breadth is demonstrated by creating models of DNA replication dynamics, the gene expression dynamics in response to DNA methylation damage, and a multisite phosphorylation switch. The flexibility of these models is shown by adapting the DNA replication model to further include two topics of interest from the literature: cooperative origin firing and replication fork barriers. The Beacon Calculus is supported with the open-source simulator bcs (https://github.com/MBoemo/bcs.git) to allow users to develop and simulate their own models.
Asunto(s)
Biología Computacional/métodos , Modelos Biológicos , Modelos Teóricos , Simulación por Computador , Daño del ADN/fisiología , Replicación del ADN/fisiología , FosforilaciónRESUMEN
Centromeres play several important roles in ensuring proper chromosome segregation. Not only do they promote kinetochore assembly for microtubule attachment, but they also support robust sister chromatid cohesion at pericentromeres and facilitate replication of centromeric DNA early in S phase. However, it is still elusive how centromeres orchestrate all these functions at the same site. Here, we show that the budding yeast Dbf4-dependent kinase (DDK) accumulates at kinetochores in telophase, facilitated by the Ctf19 kinetochore complex. This promptly recruits Sld3-Sld7 replication initiator proteins to pericentromeric replication origins so that they initiate replication early in S phase. Furthermore, DDK at kinetochores independently recruits the Scc2-Scc4 cohesin loader to centromeres in G1 phase. This enhances cohesin loading and facilitates robust pericentromeric cohesion in S phase. Thus, we have found the central mechanism by which kinetochores orchestrate early S phase DNA replication and robust sister chromatid cohesion at microtubule attachment sites.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Cinetocoros/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Centrómero/genética , Centrómero/metabolismo , Cromátides/genética , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Fase S/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
DNA replication is a stochastic process with replication forks emanating from multiple replication origins. The origins must be licenced in G1, and the replisome activated at licenced origins in order to generate bi-directional replication forks in S-phase. Differential firing times lead to origin interference, where a replication fork from an origin can replicate through and inactivate neighbouring origins (origin obscuring). We developed a Bayesian algorithm to characterize origin firing statistics from Okazaki fragment (OF) sequencing data. Our algorithm infers the distributions of firing times and the licencing probabilities for three consecutive origins. We demonstrate that our algorithm can distinguish partial origin licencing and origin obscuring in OF sequencing data from Saccharomyces cerevisiae and human cell types. We used our method to analyse the decreased origin efficiency under loss of Rat1 activity in S. cerevisiae, demonstrating that both reduced licencing and increased obscuring contribute. Moreover, we show that robust analysis is possible using only local data (across three neighbouring origins), and analysis of the whole chromosome is not required. Our algorithm utilizes an approximate likelihood and a reversible jump sampling technique, a methodology that can be extended to analysis of other mechanistic processes measurable through Next Generation Sequencing data.
Asunto(s)
Algoritmos , Replicación del ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Origen de Réplica/genética , Teorema de Bayes , Cromosomas/genética , ADN/biosíntesis , ADN/genética , Exorribonucleasas/genética , Humanos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Procesos EstocásticosRESUMEN
Genomes are replicated in a reproducible temporal pattern. Current methods for assaying allele replication timing are time consuming and/or expensive. These include high-throughput sequencing which can be used to measure DNA copy number as a proxy for allele replication timing. Here, we use droplet digital PCR to study DNA replication timing at multiple loci in budding yeast and human cells. We establish that the method has temporal and spatial resolutions comparable to the high-throughput sequencing approaches, while being faster than alternative locus-specific methods. Furthermore, the approach is capable of allele discrimination. We apply this method to determine relative replication timing across timing transition zones in cultured human cells. Finally, multiple samples can be analysed in parallel, allowing us to rapidly screen kinetochore mutants for perturbation to centromere replication timing. Therefore, this approach is well suited to the study of locus-specific replication and the screening of cis- and trans-acting mutants to identify mechanisms that regulate local genome replication timing.
Asunto(s)
Momento de Replicación del ADN/fisiología , Replicación del ADN/genética , Reacción en Cadena de la Polimerasa/métodos , Alelos , Células Cultivadas , Variaciones en el Número de Copia de ADN/genética , Momento de Replicación del ADN/genética , Genoma Fúngico , Genoma Humano , Células HeLa , Humanos , Células Jurkat , Saccharomyces cerevisiae , Saccharomycetales/genética , Sensibilidad y EspecificidadRESUMEN
The Rif1 protein negatively regulates telomeric TG repeat length in the budding yeast Saccharomyces cerevisiae, but how it prevents telomere over-extension is unknown. Rif1 was recently shown to control DNA replication by acting as a Protein Phosphatase 1 (PP1)-targeting subunit. Therefore, we investigated whether Rif1 controls telomere length by targeting PP1 activity. We find that a Rif1 mutant defective for PP1 interaction causes a long-telomere phenotype, similar to that of rif1Δ cells. Tethering PP1 at a specific telomere partially substitutes for Rif1 in limiting TG repeat length, confirming the importance of PP1 in telomere length control. Ablating Rif1-PP1 interaction is known to cause precocious activation of telomere-proximal replication origins and aberrantly early telomere replication. However, we find that Rif1 still limits telomere length even if late replication is forced through deletion of nearby replication origins, indicating that Rif1 can control telomere length independent of replication timing. Moreover we find that, even at a de novo telomere created after DNA synthesis during a mitotic block, Rif1-PP1 interaction is required to suppress telomere lengthening and prevent inappropriate recruitment of Tel1 kinase. Overall, our results show that Rif1 controls telomere length by recruiting PP1 to directly suppress telomerase-mediated TG repeat lengthening.
Asunto(s)
Proteína Fosfatasa 1/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Homeostasis del Telómero , Proteínas de Unión a Telómeros/metabolismo , Momento de Replicación del ADN , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación , Proteínas Serina-Treonina Quinasas/metabolismo , Origen de Réplica , Proteínas Represoras/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Telómero/metabolismo , Proteínas de Unión a Telómeros/genéticaRESUMEN
The common ancestry of archaea and eukaryotes is evident in their genome architecture. All eukaryotic and several archaeal genomes consist of multiple chromosomes, each replicated from multiple origins. Three scenarios have been proposed for the evolution of this genome architecture: 1) mutational diversification of a multi-copy chromosome; 2) capture of a new chromosome by horizontal transfer; 3) acquisition of new origins and splitting into two replication-competent chromosomes. We report an example of the third scenario: the multi-origin chromosome of the archaeon Haloferax volcanii has split into two elements via homologous recombination. The newly generated elements are bona fide chromosomes, because each bears "chromosomal" replication origins, rRNA loci, and essential genes. The new chromosomes were stable during routine growth but additional genetic manipulation, which involves selective bottlenecks, provoked further rearrangements. To the best of our knowledge, rearrangement of a naturally evolved prokaryotic genome to generate two new chromosomes has not been described previously.
Asunto(s)
Evolución Biológica , Cromosomas de Archaea , Genoma Arqueal , Haloferax volcanii/genética , ReplicónRESUMEN
Chromosome duplication normally initiates through the assembly of replication fork complexes at defined origins. DNA synthesis by any one fork is thought to cease when it meets another travelling in the opposite direction, at which stage the replication machinery may simply dissociate before the nascent strands are finally ligated. But what actually happens is not clear. Here we present evidence consistent with the idea that every fork collision has the potential to threaten genomic integrity. In Escherichia coli this threat is kept at bay by RecG DNA translocase and by single-strand DNA exonucleases. Without RecG, replication initiates where forks meet through a replisome assembly mechanism normally associated with fork repair, replication restart and recombination, establishing new forks with the potential to sustain cell growth and division without an active origin. This potential is realized when roadblocks to fork progression are reduced or eliminated. It relies on the chromosome being circular, reinforcing the idea that replication initiation is triggered repeatedly by fork collision. The results reported raise the question of whether replication fork collisions have pathogenic potential for organisms that exploit several origins to replicate each chromosome.
Asunto(s)
Cromosomas Bacterianos/metabolismo , Replicación del ADN , Escherichia coli/genética , Origen de Réplica , Proteínas Bacterianas/metabolismo , Bacteriófagos/genética , Bacteriófagos/fisiología , Cromosomas Bacterianos/genética , ADN Helicasas/metabolismo , ADN Bacteriano , ADN Circular , ADN de Cadena Simple , Proteínas de Unión al ADN/metabolismo , Escherichia coli/enzimología , Escherichia coli/crecimiento & desarrollo , Escherichia coli/virología , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonucleasa V/metabolismo , Exonucleasas/metabolismo , Marcadores Genéticos/genética , Inestabilidad GenómicaRESUMEN
DNA replication initiates at defined sites called origins, which serve as binding sites for initiator proteins that recruit the replicative machinery. Origins differ in number and structure across the three domains of life and their properties determine the dynamics of chromosome replication. Bacteria and some archaea replicate from single origins, whereas most archaea and all eukaryotes replicate using multiple origins. Initiation mechanisms that rely on homologous recombination operate in some viruses. Here we show that such mechanisms also operate in archaea. We use deep sequencing to study replication in Haloferax volcanii and identify four chromosomal origins of differing activity. Deletion of individual origins results in perturbed replication dynamics and reduced growth. However, a strain lacking all origins has no apparent defects and grows significantly faster than wild type. Origin-less cells initiate replication at dispersed sites rather than at discrete origins and have an absolute requirement for the recombinase RadA, unlike strains lacking individual origins. Our results demonstrate that homologous recombination alone can efficiently initiate the replication of an entire cellular genome. This raises the question of what purpose replication origins serve and why they have evolved.
Asunto(s)
Replicación del ADN/genética , Haloferax volcanii/crecimiento & desarrollo , Haloferax volcanii/genética , Origen de Réplica , Proteínas Arqueales/metabolismo , ADN de Archaea/análisis , ADN de Archaea/biosíntesis , ADN de Archaea/genética , Proteínas de Unión al ADN/metabolismo , Evolución Molecular , Secuenciación de Nucleótidos de Alto Rendimiento , Recombinación Homóloga/genética , Modelos Genéticos , Origen de Réplica/genética , Factores de TiempoRESUMEN
Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology.
Asunto(s)
Replicación del ADN , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Origen de Réplica , Saccharomyces cerevisiae/genéticaRESUMEN
Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as 'chromatin-dependent' when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at 'chromatin-dependent' origins. Finally, the 'chromatin-dependent' class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time.
Asunto(s)
Cromatina/genética , Replicación del ADN/genética , Complejo de Reconocimiento del Origen/genética , Saccharomyces cerevisiae/genética , Sitios de Unión , Cromosomas/genética , ADN/genética , Proteínas de Unión al ADN/genética , Fase G1/genética , Nucleosomas/genética , Unión Proteica , Origen de Réplica/genéticaRESUMEN
DNA replication initiates from defined locations called replication origins; some origins are highly active, whereas others are dormant and rarely used. Origins also differ in their activation time, resulting in particular genomic regions replicating at characteristic times and in a defined temporal order. Here we report the comparison of genome replication in four budding yeast species: Saccharomyces cerevisiae, S. paradoxus, S. arboricolus, and S. bayanus. First, we find that the locations of active origins are predominantly conserved between species, whereas dormant origins are poorly conserved. Second, we generated genome-wide replication profiles for each of these species and discovered that the temporal order of genome replication is highly conserved. Therefore, active origins are not only conserved in location, but also in activation time. Only a minority of these conserved origins show differences in activation time between these species. To gain insight as to the mechanisms by which origin activation time is regulated we generated replication profiles for a S. cerevisiae/S. bayanus hybrid strain and find that there are both local and global regulators of origin function.
Asunto(s)
Momento de Replicación del ADN , Replicación del ADN , Origen de Réplica , Saccharomyces cerevisiae/genética , Quimera/genética , Evolución Molecular , Regulación Fúngica de la Expresión Génica , Telómero/genéticaRESUMEN
During S phase, the entire genome must be precisely duplicated, with no sections of DNA left unreplicated. Here, we develop a simple mathematical model to describe the probability of replication failing due to the irreversible stalling of replication forks. We show that the probability of complete genome replication is maximized if replication origins are evenly spaced, the largest inter-origin distances are minimized, and the end-most origins are positioned close to chromosome ends. We show that origin positions in the yeast Saccharomyces cerevisiae genome conform to all three predictions thereby maximizing the probability of complete replication if replication forks stall. Origin positions in four other yeasts-Kluyveromyces lactis, Lachancea kluyveri, Lachancea waltii and Schizosaccharomyces pombe-also conform to these predictions. Equating failure rates at chromosome ends with those in chromosome interiors gives a mean per nucleotide fork stall rate of â¼5 × 10(-8), which is consistent with experimental estimates. Using this value in our theoretical predictions gives replication failure rates that are consistent with data from replication origin knockout experiments. Our theory also predicts that significantly larger genomes, such as those of mammals, will experience a much greater probability of replication failure genome-wide, and therefore will likely require additional compensatory mechanisms.
Asunto(s)
Genoma Fúngico , Origen de Réplica , Saccharomyces cerevisiae/genética , Cromosomas Fúngicos , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Modelos Genéticos , Complejos Multienzimáticos/metabolismo , Saccharomyces cerevisiae/metabolismo , Levaduras/genéticaRESUMEN
Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis.
Asunto(s)
Proteínas de Ciclo Celular/genética , Gametogénesis/genética , Plasmodium malariae/genética , Proteínas de Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Proteínas Cdc20 , Proteínas Cdh1 , Genes Protozoarios/fisiología , Células Germinativas/metabolismo , Células Germinativas/fisiología , Cinetocoros/metabolismo , Cinetocoros/fisiología , Malaria/parasitología , Masculino , Ratones , Datos de Secuencia Molecular , Organismos Modificados Genéticamente , Filogenia , Plasmodium malariae/crecimiento & desarrollo , Plasmodium malariae/metabolismo , Plasmodium malariae/fisiología , Homología de SecuenciaRESUMEN
OriDB (http://www.oridb.org/) is a database containing collated genome-wide mapping studies of confirmed and predicted replication origin sites. The original database collated and curated Saccharomyces cerevisiae origin mapping studies. Here, we report that the OriDB database and web site have been revamped to improve user accessibility to curated data sets, to greatly increase the number of curated origin mapping studies, and to include the collation of replication origin sites in the fission yeast Schizosaccharomyces pombe. The revised database structure underlies these improvements and will facilitate further expansion in the future. The updated OriDB for S. cerevisiae is available at http://cerevisiae.oridb.org/ and for S. pombe at http://pombe.oridb.org/.