RESUMO
The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.
Assuntos
Autoantígenos/genética , Proteínas Cromossômicas não Histona/genética , Histonas/genética , Autoantígenos/metabolismo , Centrômero , Proteína Centromérica A , Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Humanos , Cinetocoros , Escleroderma Sistêmico/genética , Terminologia como AssuntoRESUMO
On monocentric chromosomes the centromere is the chromosomal site at which the kinetochore complex is assembled. This complex mediates the attachment and movement of chromosomes along spindle microtubules. The centromere is usually the last site to retain cohesion between sister centromeres. The location of the main sensor for defective spindle assembly at the kinetochore allows the release of this cohesion, and thus progression through mitosis, to be held in check until key events have been completed. The intricate nature of the centromere-kinetochore complexes and the events they co-ordinate and react to is presently being dissected by studies in several organisms. In particular, several new kinetochore proteins have been identified in many organisms over the last year.
Assuntos
Centrômero/fisiologia , Cromossomos/fisiologia , Adesividade , Animais , Cromatina/fisiologia , Proteínas Fúngicas/fisiologia , Humanos , Cinetocoros/fisiologia , Cinetocoros/ultraestrutura , Meiose/fisiologia , Microtúbulos/fisiologia , Mitose/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Schizosaccharomyces/citologia , Schizosaccharomyces/fisiologiaRESUMO
Centromeres are heterochromatic in many organisms, but the mitotic function of this silent chromatin remains unknown. During cell division, newly replicated sister chromatids must cohere until anaphase when Scc1/Rad21-mediated cohesion is destroyed. In metazoans, chromosome arm cohesins dissociate during prophase, leaving centromeres as the only linkage before anaphase. It is not known what distinguishes centromere cohesion from arm cohesion. Fission yeast Swi6 (a Heterochromatin protein 1 counterpart) is a component of silent heterochromatin. Here we show that this heterochromatin is specifically required for cohesion between sister centromeres. Swi6 is required for association of Rad21-cohesin with centromeres but not along chromosome arms and, thus, acts to distinguish centromere from arm cohesion. Therefore, one function of centromeric heterochromatin is to attract cohesin, thereby ensuring sister centromere cohesion and proper chromosome segregation.
Assuntos
Proteínas de Ciclo Celular , Centrômero/metabolismo , Proteínas Fúngicas/metabolismo , Heterocromatina/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Fatores de Transcrição/metabolismo , Centrômero/fisiologia , Cromátides/metabolismo , Proteínas Cromossômicas não Histona , Segregação de Cromossomos , Cromossomos Fúngicos/metabolismo , Proteínas Fúngicas/genética , Hibridização in Situ Fluorescente , Metáfase , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genética , Transcrição Gênica , CoesinasRESUMO
An emerging view is that the formation of active centromeres is modulated in an epigenetic manner reflecting the association of centromeres with heterochromatin. Support for this comes from studies on fission yeast centromeres, the properties of human neocentromeres and dicentric chromosomes, and analyses of Drosophila minichromosome deletion derivatives. A link has been established between tension across kinetochores and the phosphorylation status of kinetochore components. Vertebrate homologues of yeast MAD2 have recently been isolated and localized to kinetochores, indicating that components of the spindle integrity checkpoint are conserved. The linkage between sister chromatids is only dissolved at anaphase during mitotic and meiotic divisions. Phenotypic and localization data combined with their pattern of rapid degradation at anaphase have implicated several yeast and Drosophila proteins in aspects of sister chromatid cohesion.
Assuntos
Centrômero , Cromatina , Anáfase , Animais , Cromossomos Fúngicos , Heterocromatina , Humanos , Meiose , Schizosaccharomyces , Transdução de SinaisRESUMO
BACKGROUND: Proteins such as HP1, found in fruit flies and mammals, and Swi6, its fission yeast homologue, carry a chromodomain (CD) and a chromo shadow domain (CSD). These proteins are required to form functional transcriptionally silent centromeric chromatin, and their mutation leads to chromosome segregation defects. CSDs have only been found in tandem in proteins containing the related CD. Most HP1-interacting proteins have been found to associate through the CSD and many of these ligands contain a conserved pentapeptide motif. RESULTS: The 1.9 A crystal structure of the Swi6 CSD is presented here. This reveals a novel dimeric structure that is distinct from the previously reported monomeric nuclear magnetic resonance (NMR) structure of the CD from the mouse modifier 1 protein (MoMOD1, also known as HP1beta or M31). A prominent pit with a non-polar base is generated at the dimer interface, and is commensurate with binding an extended pentapeptide motif. Sequence alignments based on this structure highlight differences between CDs and CSDs that are superimposed on a common structural core. The analyses also revealed a previously unrecognised circumferential hydrophobic sash around the surface of the CD structure. CONCLUSIONS: Dimerisation through the CSD of HP1-like proteins results in the simultaneous formation of a putative protein-protein interaction pit, providing a potential means of targeting CSD-containing proteins to particular chromatin sites.
Assuntos
Proteínas Fúngicas/química , Conformação Proteica , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Homólogo 5 da Proteína Cromobox , Dimerização , Proteínas Fúngicas/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Dobramento de Proteína , Schizosaccharomyces/química , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismoRESUMO
The ends of human chromosomes have been shown recently to resemble those of simple organisms. With this in mind, we discuss the nature and possible significance of rare chromosome fusion events thought to involve telomeres, particularly those fusion events found in some tumours. Also we argue that interstitial telomere-like stretches may be particularly prone to recombination, breakage and fragility.
Assuntos
Cromossomos Humanos , Sequências Repetitivas de Ácido Nucleico , Animais , Replicação do DNA , Humanos , Masculino , Neoplasias/genética , Recombinação GenéticaRESUMO
The centromere is required to ensure the equal distribution of replicated chromosomes to daughter nuclei. Centromeres are frequently associated with heterochromatin, an enigmatic nuclear component that causes the epigenetic transcriptional repression of nearby marker genes (position-effect variegation or silencing). The process of chromosome segregation by movement along microtubules to spindle poles is highly conserved, yet the putative cis-acting centromeric DNA sequences bear little or no similarity across species. Recently, studies in several systems have revealed that the centromere itself might be epigenetically regulated and that the higher-order structure of the underlying heterochromatin contributes to centromere function and kinetochore assembly.
Assuntos
Centrômero/fisiologia , Células Eucarióticas/fisiologia , Modelos Genéticos , Animais , Sequência de Bases , Cromatina/química , Cromatina/genética , Sequência Conservada , Drosophila/genética , Humanos , Cinetocoros/metabolismo , Splicing de RNA , Leveduras/genéticaRESUMO
Tf1 is a long terminal repeat (LTR)-containing retrotransposon that propagates within the fission yeast Schizosaccharomyces pombe. LTR-retrotransposons possess significant similarity to retroviruses and therefore serve as retrovirus models. To determine what features of the host cell are important for the proliferation of this class of retroelements, we screened for mutations in host genes that reduced the transposition activity of Tf1. We report here the isolation and characterization of pst1(+), a gene required for Tf1 transposition. The predicted amino acid sequence of Pst1p possessed high sequence homology with the Sin3 family of proteins, known for their interaction with histone deacetylases. However, unlike the SIN3 gene of Saccharomyces cerevisiae, pst1(+) is essential for cell viability. Immunofluorescence microscopy indicated that Pst1p was localized in the nucleus. Consistent with the critical role previously reported for Sin3 proteins in the histone acetylation process, we found that the growth of the strain with the pst1-1 allele was supersensitive to the specific histone deacetylase inhibitor trichostatin A. However, our analysis of strains with the pst1-1 mutation was unable to detect any changes in the acetylation of specific lysines of histones H3 and H4 as measured in bulk chromatin. Interestingly, the pst1-1 mutant strain produced wild-type levels of Tf1-encoded proteins and cDNA, indicating that the defect in transposition occurred after reverse transcription. The results of immunofluorescence microscopy showed that the nuclear localization of the Tf1 capsid protein was disrupted in the strain with the pst1-1 mutation, indicating an important role of pst1(+) in modulating the nuclear import of Tf1 virus-like particles.
Assuntos
Proteínas Fúngicas/genética , Proteínas Repressoras/genética , Retroelementos , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/metabolismo , Hemaglutininas , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Dados de Sequência Molecular , Mutagênese , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe , Fatores de Transcrição/metabolismoRESUMO
Although silencing is a significant form of transcriptional regulation, the functional and mechanistic limits of its conservation have not yet been established. We have identified the Schizosaccharomyces pombe hst4(+) gene as a member of the SIR2/HST silencing gene family that is defined in organisms ranging from bacteria to humans. hst4Delta mutants grow more slowly than wild-type cells and have abnormal morphology and fragmented DNA. Mutant strains show decreased silencing of reporter genes at both telomeres and centromeres. hst4(+) appears to be important for centromere function as well because mutants have elevated chromosome-loss rates and are sensitive to a microtubule-destabilizing drug. Consistent with a role in chromatin structure, Hst4p localizes to the nucleus and appears concentrated in the nucleolus. hst4Delta mutant phenotypes, including growth and silencing phenotypes, are similar to those of the Saccharomyces cerevisiae HSTs, and at a molecular level, hst4(+) is most similar to HST4. Furthermore, hst4(+) is a functional homologue of S. cerevisiae HST3 and HST4 in that overexpression of hst4(+) rescues the temperature-sensitivity and telomeric silencing defects of an hst3Delta hst4Delta double mutant. These results together demonstrate that a SIR-like silencing mechanism is conserved in the distantly related yeasts and is likely to be found in other organisms from prokaryotes to mammals.
Assuntos
Centrômero/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Inativação Gênica , Histona Desacetilases , Schizosaccharomyces/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Transativadores/genética , Clonagem Molecular , Proteínas de Ligação a DNA/química , Imunofluorescência , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Microscopia de Contraste de Fase , Mutação , Fenótipo , Saccharomyces cerevisiae/genética , Schizosaccharomyces/crescimento & desenvolvimento , Alinhamento de Sequência , Sirtuína 2 , Sirtuínas , Telômero/genética , Transativadores/químicaRESUMO
Linearized bovine papillomavirus type 1 (BPV-1) DNA was introduced into mouse C127 cells, where it recircularized and replicated as an intact monomeric, extrachromosomal circular form in the resulting transformants. These cells contained a mixture of complex high molecular weight forms that were converted to a linear form of approximately BPV-1 size upon digestion with an enzyme that cuts once within the BPV-1 genome. Further analysis of one of these cell lines revealed that these high molecular weight forms consisted of two components. One was detected on agarose gels as a diffuse smear of slow-migrating material representing linear forms that were tightly associated with host chromosomes, probably by integration. The second component was composed of discrete-sized oligomeric open and supercoiled extrachromosomal circular forms of up to approximately 48 X 10(3) base-pairs (6 tandemly linked BPV-1 genomes) in size. No catenated (interlocked) forms could be detected.
Assuntos
Papillomavirus Bovino 1/genética , DNA Viral , Papillomaviridae/genética , Transformação Genética , Animais , Autorradiografia , Papillomavirus Bovino 1/metabolismo , Linhagem Celular , Centrifugação com Gradiente de Concentração , DNA Viral/metabolismo , Eletroforese em Gel de Ágar , Exonucleases/metabolismo , Camundongos , Mitose , Hibridização de Ácido NucleicoRESUMO
In the fission yeast Schizosaccharomyces pombe genes are transcriptionally silenced when placed within centromeres, within or close to the silent mating-type loci or adjacent to telomeres. Factors required to maintain mating-type silencing also affect centromeric silencing and chromosome segregation. We isolated mutations that alleviate repression of marker genes in the inverted repeats flanking the central core of centromere I. Mutations csp1 to 13 (centromere: suppressor of position effect) defined 12 loci. Ten of the csp mutants have no effect on mat2/3 or telomere silencing. All csp mutants allow some expression of genes in the centromeric flanking repeat, but expression in the central core is undetectable. Consistent with defective centromere structure and function, chromosome loss rates are elevated in all csp mutants. Mutants csp1 to 6 are temperature-sensitive lethal and csp3 and csp6 cells are defective in mitosis at 36 degrees. csp7 to 13 display a high incidence of lagging chromosomes on late anaphase spindles. Thus, by screening for mutations that disrupt silencing in the flanking region of a fission yeast centromere a novel collection of mutants affecting centromere architecture and chromosome segregation has been isolated.
Assuntos
Centrômero/genética , Cromossomos Fúngicos/genética , Schizosaccharomyces/genética , Transcrição Gênica , DNA Fúngico/genética , Eletroforese em Gel de Campo Pulsado , Regulação Fúngica da Expressão Gênica , Marcadores Genéticos , Genótipo , Mutagênese , RNA Fúngico/genética , Sequências Repetitivas de Ácido Nucleico , Schizosaccharomyces/citologia , Schizosaccharomyces/crescimento & desenvolvimento , Supressão GenéticaRESUMO
Various recombinant constructions involving bovine papillomavirus type 1 (BPV-1) DNA and bacterial plasmids have been tested for their ability to transform mouse C127 cells and replicate as intact extrachromosomal monomeric structures. When BPV-1 DNA was linked to pBR328, pAT153 or derivatives of these plasmids lacking the 344 bp HindIII-BamHI fragment or another small segment, the resulting vectors replicated in C127 cells as high molecular weight structures and, in some cases, deleted extrachromosomal forms. The sequences which became deleted were generally the non-BPV-1 sequences. Duplication of the 3' distal enhancer sequence of BPV-1 DNA in one of the vectors increased its stability upon introduction into C127 cells, but some deleted and high molecular weight forms were still observed.
Assuntos
Papillomavirus Bovino 1/genética , DNA Viral/genética , Elementos Facilitadores Genéticos , Genes Reguladores , Vetores Genéticos , Papillomaviridae/genética , Plasmídeos , Animais , Linhagem Celular , Transformação Celular Viral , Replicação do DNA , DNA Viral/análise , Escherichia coli/genética , Camundongos , TransfecçãoRESUMO
RNA interference (RNAi) is a conserved silencing mechanism whereby double-strand RNA induces specific down-regulation of homologous sequences. In the fission yeast Schizosaccharomyces pombe, centromeric heterochromatin assembly is an RNAi-dependent process. Noncoding RNAs transcribed from pericentromeric repeat sequences are processed into short interfering RNAs (siRNAs) that direct the Argonaute-containing RNA-induced transcriptional silencing (RITS) effector complex to homologous nascent transcripts. RITS is required for H3K9 methylation by the histone methyltransferase (HMT) Clr4; conversely, H3K9 methylation can attract RITS to chromatin via binding of the chromodomain protein Chp1. This codependency has hampered dissection of the order of events and mechanisms of cross talk between the RNAi and chromatin modification machineries. To tackle this problem, we have developed systems that reconstitute heterochromatin at a euchromatic locus, using either hairpin triggers or DNA-tethered chromatin-modifying complexes. These systems reveal that RNAi is sufficient to promote heterochromatin assembly in cis and that direct recruitment of the HMT Clr4 can bypass the role of RNAi in heterochromatin assembly. We have also characterized a new pathway component, Stc1, that translates the RNAi signal into chromatin marks. We discuss the implications of these findings for our understanding of the mechanism and function of RNAi-directed heterochromatin assembly at centromeres.
Assuntos
Centrômero/metabolismo , Heterocromatina/metabolismo , Interferência de RNA , Heterocromatina/química , Conformação de Ácido Nucleico , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismoAssuntos
Cromossomos Fúngicos , Clonagem Molecular/métodos , DNA Recombinante/biossíntese , Vetores Genéticos , Schizosaccharomyces/genética , Transformação Genética , DNA Recombinante/análise , Eletroforese em Gel de Ágar/métodos , Indicadores e Reagentes , Cariotipagem , Cinética , Lipossomos , Fosfatidiletanolaminas/farmacologia , Plasmídeos , Protoplastos/fisiologia , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Transformação Genética/efeitos dos fármacosAssuntos
Cromossomos Humanos/ultraestrutura , Técnicas Genéticas , Telômero/ultraestrutura , Sequência de Bases , Cromossomos Artificiais de Levedura , Clonagem Molecular , DNA/genética , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Vetores Genéticos , Humanos , Sequências Repetitivas de Ácido Nucleico , Saccharomyces cerevisiae/genética , Esferoplastos/ultraestrutura , Transformação GenéticaRESUMO
The efficiency of transformation of Schizosaccharomyces pombe has been increased 10- to 50-fold over previously reported methods. By using 1 microgram of plasmid, 7.0 x 10(5) transformants are regularly obtained. This increased transformation efficiency is mainly due to the inclusion of the cationic liposome-forming reagent Lipofectin in the protocol. Various parameters affecting transformation of Sc. pombe in the presence of Lipofectin have been examined. Lipofectin can also be used to increase transformation efficiency in Saccharomyces cerevisiae. It is also demonstrated that by using this improved transformation procedure, linear minichromosomes of greater than 500 kilobases can be introduced into Sc. pombe with relative ease. These minichromosomes can replicate as stable linear molecules upon reintroduction into Sc. pombe, demonstrating that Sc. pombe telomeres retain function when reintroduced as naked DNA. The ability of Sc. pombe to admit large DNA molecules indicates that it should be feasible to clone large DNA from other organisms in Sc. pombe.
Assuntos
Cromossomos Fúngicos/ultraestrutura , Saccharomycetales/genética , Schizosaccharomyces/genética , Transformação Genética , DNA Recombinante , Eletroforese em Gel de Ágar , Vetores Genéticos , Peso Molecular , Fosfatidiletanolaminas/farmacologia , Plasmídeos , Transformação Genética/efeitos dos fármacosRESUMO
The investigation of fission yeast chromosome structure and function has moved rapidly over the past 10 years. The isolation of replication origins, telomeres and centromeres has allowed the development of minichromosomes, a yeast artificial chromosome (YAC)-like cloning system and investigations into chromosome segregation and behaviour during mitosis and meiosis. Many mutants have been isolated which are defective in chromosome segregation. The development of the fluorescent in-situ hybridization (FISH) technique for use in S. pombe has allowed the localization of centromeres and telomeres throughout mitosis and meiosis. In combination with indirect immunofluorescence to detect spindle and chromosomal proteins, the FISH technique should further advance our understanding of fission yeast chromosome structure and function. The recent discovery of a heterochromatin-like structure mediating transcriptional repression at centromeres reinforces the notion that fission yeast centromeres are similar to those of larger eukaryotes. Further characterization of such phenomena will accelerate the genetic dissection of this important chromosomal element.
Assuntos
Cromossomos Fúngicos , Schizosaccharomyces/genética , Sequência de Bases , Divisão Celular , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Cromossomos Fúngicos/fisiologia , Cromossomos Fúngicos/ultraestrutura , DNA Fúngico/análise , Heterocromatina/ultraestrutura , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico , Origem de Replicação , Schizosaccharomyces/fisiologia , Schizosaccharomyces/ultraestrutura , Telômero/químicaRESUMO
The intracellular structure of several vectors based on BPV-1 DNA has been analyzed following transfection into mouse C127 cells by the calcium phosphate method or, for the first time, by microinjection directly into the nucleus. It is shown that the method of introduction markedly affects the fate of a BPV-1 based vector. In general, microinjection appears to do little damage to DNA and is more likely to result in a vector replicating extrachromosomally as a monomeric structure of the same size as the input DNA. The method of selection for transformed cells, e.g., focus formation versus resistance to the neomycin analog G418, can also affect the intracellular state of the BPV-1 vector DNA. The nature of the recipient mammalian cell also influences whether a vector can replicate extrachromosomally or whether it integrates. BPV-1 based vectors, which replicated predominantly as multicopy intact extrachromosomal forms in mouse C127 cells, were always found to have integrated at low copy number in mouse LtAp20 cells.
Assuntos
Papillomavirus Bovino 1/genética , Vetores Genéticos , Papillomaviridae/genética , Transfecção , Animais , Células Cultivadas , DNA Viral/genética , Métodos , Camundongos , MicroinjeçõesRESUMO
The sequence requirements for in vivo telomere function in the fission yeast, Schizosaccharomyces pombe, have been investigated. A 258 bp tract of previously characterized cloned fission yeast terminal repeats adjacent to 800 bp of telomere-associated sequences is sufficient to seed new telomeres onto linearized ars-containing plasmids when introduced into cells. The resulting transformants contain unrearranged, acentric, linear episomes. Cloned telomeres, with and without telomere-associated sequences adjacent to the 258 bp terminal repeats, were utilized to introduce chromosome breaks at specific sites in a non-essential minichromosome. Truncated minichromosome derivatives were recovered containing the ura4 or ade6 gene adjacent to a newly formed telomere. These telomeres exert reversible position effects on the expression of the adjacent ura4 or ade6 genes.
Assuntos
Aberrações Cromossômicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Schizosaccharomyces/genética , Adenina/metabolismo , Divisão Celular/efeitos dos fármacos , DNA Recombinante/genética , Eletroforese em Gel de Campo Pulsado , Herança Extracromossômica , Rearranjo Gênico , Leucina/metabolismo , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , RNA Fúngico/análise , Schizosaccharomyces/crescimento & desenvolvimento , Uracila/metabolismoRESUMO
Using a combination of different oligonucleotides and restriction enzymes we have examined the gross organisation of repeats within the most distal region of human chromosomes. We demonstrate here that human telomeres do not contain a pure uniform 6 base pair repeat unit but that there are at least three types of repeat. These three types of repeat are present at the ends of most or all human chromosomes. The distribution of each type of repeat appears to be non-random. Each human telomere has a similar arrangement of these repeats relative to the ends of the chromosome. This could reflect differences in the functions that they perform, or might result from the mutation and correction processes occurring at human telomeres. The number of repeat units, the repeat types and arrangement differs at mouse telomeres. Analysing the change in length of the telomeric repeat region between an individuals blood and germline DNA reveals that this is due to variable amounts of the TTAGGG repeat and not the other repeat types. This organization of repeat units at human telomeres will only be confirmed upon the isolation and sequencing of full length (10-15 kb), intact human telomeres.