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
Chromosome fragments that lack centromeric DNA (structurally acentric chromosomes) are usually not inherited in mitosis and meiosis. We previously described the isolation, after irradiation of a Drosophila melanogaster mini-chromosome, of structurally acentric mini-chromosomes that display efficient mitotic and meiotic transmission despite their small size (under 300 kb) and lack of centromeric DNA. Here we report that these acentric mini-chromosomes bind the centromere-specific protein ZW10 and associate with the spindle poles in anaphase. The sequences in these acentric mini-chromosomes were derived from the tip of the X chromosome, which does not display centromere activity or localize ZW10, even when separated from the rest of the X. We conclude that the normally non-centromeric DNAs present in these acentric mini-chromosomes have acquired centromere function, and suggest that this example of 'neocentromere' formation involves appropriation of a self-propagating centromeric chromatin structure. The potential relevance of these observations to the identity, propagation and function of normal centromeres is discussed.
Assuntos
Proteínas de Ciclo Celular , Centrômero , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas de Insetos/metabolismo , Animais , Centrômero/metabolismo , Cromossomos , DNA/metabolismo , Ligação Proteica , Cromossomo XRESUMO
Centromere function requires the coordination of many processes including kinetochore assembly, sister chromatid cohesion, spindle attachment and chromosome movement. Here we show that CID, the Drosophila homologue of the CENP-A centromere-specific H3-like proteins, colocalizes with molecular-genetically defined functional centromeres in minichromosomes. Injection of CID antibodies into early embryos, as well as RNA interference in tissue-culture cells, showed that CID is required for several mitotic processes. Deconvolution fluorescence microscopy showed that CID chromatin is physically separate from proteins involved in sister cohesion (MEI-S332), centric condensation (PROD), kinetochore function (ROD, ZW10 and BUB1) and heterochromatin structure (HP1). CID localization is unaffected by mutations in mei-S332, Su(var)2-5 (HP1), prod or polo. Furthermore, the localization of POLO, CENP-meta, ROD, BUB1 and MEI-S332, but not PROD or HP1, depends on the presence of functional CID. We conclude that the centromere and flanking heterochromatin are physically and functionally separable protein domains that are required for different inheritance functions, and that CID is required for normal kinetochore formation and function, as well as cell-cycle progression.
Assuntos
Autoantígenos , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Heterocromatina/genética , Histonas/metabolismo , Cinetocoros/metabolismo , Animais , Anticorpos/farmacologia , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteína Centromérica A , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , DNA/metabolismo , Proteínas de Ligação a DNA , Drosophila melanogaster/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Histonas/genética , Histonas/imunologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Interfase/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Centromeric chromatin is uniquely marked by the centromere-specific histone CENP-A. For assembly of CENP-A into nucleosomes to occur without competition from H3 deposition, it was proposed that centromeres are among the first or last sequences to be replicated. In this study, centromere replication in Drosophila was studied in cell lines and in larval tissues that contain minichromosomes that have structurally defined centromeres. Two different nucleotide incorporation methods were used to evaluate replication timing of chromatin containing CID, a Drosophila homologue of CENP-A. Centromeres in Drosophila cell lines were replicated throughout S phase but primarily in mid S phase. However, endogenous centromeres and X-derived minichromosome centromeres in vivo were replicated asynchronously in mid to late S phase. Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase. We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase. These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.
Assuntos
Centrômero/metabolismo , Cromatina/metabolismo , Replicação do DNA , Proteínas de Drosophila , Histonas/metabolismo , Animais , Células Cultivadas , Centrômero/ultraestrutura , Proteína Centromérica A , Cromossomos/ultraestrutura , Proteínas de Ligação a DNA , Drosophila , Modelos Genéticos , Fase SRESUMO
The chromosomal requirements for achiasmate (nonexchange) homolog disjunction in Drosophila female meiosis I have been identified with the use of a series of molecularly defined minichromosome deletion derivatives. Efficient disjunction requires 1000 kilobases of overlap in the centric heterochromatin and is not affected by homologous euchromatin or overall size differences. Disjunction efficiency decreases linearly as heterochromatic overlap is reduced from 1000 to 430 kilobases of overlap. Further observations, including rescue experiments with nod kinesin-like protein transgenes, demonstrate that heterochromatin does not act solely to promote chromosome movement or spindle attachment. Thus, it is proposed that centric heterochromatin contains multiple pairing elements that act additively to initiate or maintain the proper alignment of achiasmate chromosomes in meiosis I. How heterochromatin could act to promote chromosome pairing is discussed here.
Assuntos
Cromossomos/fisiologia , Proteínas de Drosophila , Heterocromatina/fisiologia , Meiose , Animais , Animais Geneticamente Modificados , Deleção Cromossômica , Drosophila , Feminino , Genes de Insetos , Cinesinas , Proteínas dos Microtúbulos/genética , Não Disjunção GenéticaRESUMO
The phenomenon of position-effect variegation has long been used as evidence for the importance of chromosome position to gene expression in eukaryotes. Investigations published within the past few years demonstrate that position-effect variegation is caused by multiple mechanisms, and that direct tests of hypotheses are possible with numerous model systems.
Assuntos
Regulação da Expressão Gênica , Heterocromatina , Animais , Centrômero , Cromatina/fisiologia , DNA , Drosophila/genética , Humanos , TelômeroRESUMO
Centromeres and the associated kinetochores are involved in essential aspects of chromosome transmission. Recent advances have included the identification and understanding of proteins that have a pivotal role in centromere structure, kinetochore formation, and the coordination of chromosome inheritance with the cell cycle in several organisms. A picture is beginning to emerge of the centromere-kinetechore as a complex and dynamic structure with conservation of function at the protein level across diverse species.
Assuntos
Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Animais , Ciclo Celular/genética , Microtúbulos/metabolismoRESUMO
Attachment, or cohesion, between sister chromatids is essential for their proper segregation in mitosis and meiosis [1,2]. Sister chromatids are tightly apposed at their centromeric regions, but it is not known whether this is due to cohesion at the functional centromere or at flanking centric heterochromatin. The Drosophila MEI-S332 protein maintains sister-chromatid cohesion at the centromeric region [3]. By analyzing MEI-S332's localization requirements at the centromere on a set of minichromosome derivatives [4], we tested the role of heterochromatin and the relationship between cohesion and kinetochore formation in a complex centromere of a higher eukaryote. The frequency of MEI-S332 localization is decreased on minichromosomes with compromised inheritance, despite the consistent presence of two kinetochore proteins. Furthermore, MEI-S332 localization is not coincident with kinetochore outer-plate proteins, suggesting that it is located near the DNA. We conclude that MEI-S332 localization is driven by the functional centromeric chromatin, and binding of MEI-S332 is regulated independently of kinetochore formation. These results suggest that in higher eukaryotes cohesion is controlled by the functional centromere, and that, in contrast to yeast [5], the requirements for cohesion are separable from those for kinetochore assembly.
Assuntos
Proteínas de Ciclo Celular , Centrômero/metabolismo , Cromátides/fisiologia , Proteínas de Drosophila , Drosophila/genética , Proteínas de Insetos/metabolismo , Cinetocoros/metabolismo , Animais , Segregação de Cromossomos , Drosophila/fisiologiaRESUMO
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
We investigated whether single P element insertional mutagenesis could be used to analyze heterochromatin within the Drosophila minichromosome Dp1187. Forty-five insertions of the P[lacZ,rosy+] element onto Dp1187 (recovered among 7,825 transpositions) were highly clustered. None was recovered in centromeric heterochromatin, but 39 occurred about 40 kb from the distal telomere within a 4.7-kb hotspot containing tandem copies of a novel 1.8-kb repetitive DNA sequence. The DNA within and distal to this region lacked essential genes and displayed several other properties characteristic of heterochromatin. The rosy+ genes within the inserted transposons were inhibited by position-effect variegation, and the subtelomeric region was underrepresented in polytene salivary gland cells. These experiments demonstrated that P elements preferentially transpose into a small subset of heterochromatic sites, providing a versatile method for studying the structure and function of these chromosome regions. This approach revealed that a Drosophila chromosome contains a large region of subtelomeric heterochromatin with specific structural and genetic properties.
Assuntos
Cromossomos/ultraestrutura , Drosophila/genética , Heterocromatina , Animais , Sequência de Bases , Elementos de DNA Transponíveis , Feminino , Expressão Gênica , Masculino , Dados de Sequência Molecular , Mutagênese InsercionalRESUMO
The centromere is essential for proper segregation and inheritance of genetic information. Centromeres are generally regulated to occur exactly once per chromosome; failure to do so leads to chromosome loss or damage and loss of linked genetic material. The mechanism for faithful regulation of centromere activity and number is unknown. The presence of ectopic centromeres (neocentromeres) has allowed us to probe the requirements and characteristics of centromere activation, maintenance, and structure. We utilized chromosome derivatives that placed a 290-kilobase "test segment" in three different contexts within the Drosophila melanogaster genome--immediately adjacent to (1) centromeric chromatin, (2) centric heterochromatin, or (3) euchromatin. Using irradiation mutagenesis, we freed this test segment from the source chromosome and genetically assayed whether the liberated "test fragment" exhibited centromere activity. We observed that this test fragment behaved differently with respect to centromere activity when liberated from different chromosomal contexts, despite an apparent sequence identity. Test segments juxtaposed to an active centromere produced fragments with neocentromere activity, whereas test segments far from centromeres did not. Once established, neocentromere activity was stable. The imposition of neocentromere activity on juxtaposed DNA supports the hypothesis that centromere activity and identity is capable of spreading and is regulated epigenetically.
Assuntos
Centrômero/fisiologia , Drosophila/metabolismo , Drosophila/ultraestrutura , Animais , Southern Blotting , Centrômero/efeitos da radiação , Cromatina/metabolismo , Cromossomos/metabolismo , Cromossomos/efeitos da radiação , Cruzamentos Genéticos , DNA/metabolismo , Feminino , Raios gama , Masculino , Modelos Genéticos , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Position effect variegation (PEV) is the clonal inactivation of euchromatic or heterochromatic genes that are abnormally positioned within a chromosome. PEV can be influenced by modifiers in trans, including single gene mutations and the total amount of heterochromatin present in the genome. Terminal deletions of a Drosophila minichromosome (Dp1187) dramatically increase PEV of a yellow+ body-color gene located in cis, even when the terminal break is > 100 kb distal to the yellow gene. Here we demonstrate that terminal deficiency-associated PEV can be suppressed by the presence of a second minichromosome, a novel phenomenon termed "trans-suppression." The chromosomal elements responsible for trans-suppression were investigated using a series of minichromosomes with molecularly characterized deletions and inversions. The data suggest that trans-suppression does not involve communication between transcriptional regulatory elements on the homologues, a type of transvection known to act at the yellow locus. Furthermore, trans-suppression is not accomplished by titration through the addition of extra centric heterochromatin, a general mechanism for PEV suppression. We demonstrate that trans-suppression is disrupted by significant changes in the structure of the suppressing minichromosome, including deletions of the yellow region and centric heterochromatin, and large inversions of the centric heterochromatin. We conclude that chromosome pairing plays an important role in trans-suppression and discuss the possibility that terminal deficiency-associated PEV and trans-suppression reflect changes in nuclear positioning of the chromosomes and the gene, and/or the activity and distribution of telomere-binding proteins.
Assuntos
Deleção Cromossômica , Proteínas de Drosophila , Drosophila/genética , Proteínas de Insetos/genética , Supressão Genética , Animais , HeterocromatinaRESUMO
Heterochromatin is a ubiquitous yet poorly understood component of multicellular eukaryotic genomes. Major gaps exist in our knowledge of the nature and overall organization of DNA sequences present in heterochromatin. We have investigated the molecular structure of the 1 Mb of centric heterochromatin in the Drosophila minichromosome Dp1187. A genetic screen of irradiated minichromosomes yielded rearranged derivatives of Dp1187 whose structures were determined by pulsed-field Southern analysis and PCR. Three Dp1187 deletion derivatives and an inversion had one breakpoint in the euchromatin and one in the heterochromatin, providing direct molecular access to previously inaccessible parts of the heterochromatin. End-probed pulsed-field restriction mapping revealed the presence of at least three "islands" of complex DNA, Tahiti, Moorea, and Bora Bora, constituting approximately one half of the Dp1187 heterochromatin. Pulsed-field Southern analysis demonstrated that Drosophila heterochromatin in general is composed of alternating blocks of complex DNA and simple satellite DNA. Cloning and sequencing of a small part of one island, Tahiti, demonstrated the presence of a retroposon. The implications of these findings to heterochromatin structure and function are discussed.
Assuntos
DNA/química , Drosophila/genética , Heterocromatina/genética , Animais , Sequência de Bases , Cromossomos , DNA Satélite , Feminino , Masculino , Dados de Sequência Molecular , Mapeamento por Restrição , RetroelementosRESUMO
Two different schemes were used to demonstrate that Drosophila P elements preferentially transpose into genomic regions close to their starting sites. A starting element with weak rosy+ marker gene expression was mobilized from its location in the subtelomeric region of the 1,300-kb Dp1187 minichromosome. Among progeny lines with altered rosy+ expression, a much higher than expected frequency contained new insertions on Dp1187. Terminal deficiencies were also recovered frequently. In a second screen, a rosy(+)-marked element causing a lethal mutation of the cactus gene was mobilized in male and female germlines, and viable revertant chromosomes were recovered that still contained a rosy+ gene due to an intrachromosomal transposition. New transpositions recovered using both methods were mapped between 0 and 128 kb from the starting site. Our results suggested that some mechanism elevates the frequency 43-67-fold with which a P element inserts near its starting site. Local transposition is likely to be useful for enhancing the rate of insertional mutation within predetermined regions of the genome.
Assuntos
Cromossomos , Elementos de DNA Transponíveis , Drosophila melanogaster/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Elementos de DNA Transponíveis/genética , Feminino , Genes Letais , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Mutagênese Insercional , Recombinação Genética , Deleção de SequênciaRESUMO
Deletions in the Drosophila minichromosome Dp1187 were used to investigate the genetic interactions of trans-acting genes with the centromere. Mutations in several genes known to have a role in chromosome inheritance were shown to have dominant effects on the stability of minichromosomes with partially defective centromeres. Heterozygous mutations in the ncd and klp3A kinesin-like protein genes strongly reduced the transmission of minichromosomes missing portions of the genetically defined centromere but had little effect on the transmission of minichromosomes with intact centromeres. Using this approach, ncd and klp3A were shown to require only the centromeric region of the chromosome for their roles in chromosome segregation. Increased gene dosage also affected minichromosome transmission and was used to demonstrate that the nod kinesin-like protein gene interacts genetically with the centro mere, in addition to interacting with extracentromeric regions as demonstrated previously. The results presented in this study strongly suggest that dominant genetic interactions between mutations and centromere-defective minichromosomes could be used effectively to identify novel genes necessary for centromere function.
Assuntos
Centrômero/fisiologia , Cromossomos , Proteínas de Drosophila , Drosophila melanogaster/genética , Transativadores/genética , Animais , Dosagem de Genes , Heterozigoto , Cinesinas , Proteínas dos Microtúbulos/genética , MutaçãoRESUMO
Faithful chromosome inheritance is a fundamental biological activity and errors contribute to birth defects and cancer progression. We have performed a P-element screen in Drosophila melanogaster with the aim of identifying novel candidate genes involved in inheritance. We used a "sensitized" minichromosome substrate (J21A) to screen approximately 3,000 new P-element lines for dominant effects on chromosome inheritance and recovered 78 Sensitized chromosome inheritance modifiers (Scim). Of these, 69 decreased minichromosome inheritance while 9 increased minichromosome inheritance. Fourteen mutations are lethal or semilethal when homozygous and all exhibit dramatic mitotic defects. Inverse PCR combined with genomic analyses identified P insertions within or close to genes with previously described inheritance functions, including wings apart-like (wapl), centrosomin (cnn), and pavarotti (pav). Further, lethal insertions in replication factor complex 4 (rfc4) and GTPase-activating protein 1 (Gap1) exhibit specific mitotic chromosome defects, discovering previously unknown roles for these proteins in chromosome inheritance. The majority of the lines represent mutations in previously uncharacterized loci, many of which have human homologs, and we anticipate that this collection will provide a rich source of mutations in new genes required for chromosome inheritance in metazoans.
Assuntos
Proteínas do Citoesqueleto , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insetos , Proteínas dos Microfilamentos , Proteínas de Ligação a RNA , Fatores de Transcrição , Animais , Quinase 1 do Ponto de Checagem , Elementos de DNA Transponíveis , Fator de Iniciação 4E em Eucariotos , Proteínas do Olho/genética , Feminino , Glicoproteínas/genética , Histonas/genética , Proteínas de Homeodomínio/genética , Proteínas de Insetos/genética , Masculino , Glicoproteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Fatores de Iniciação de Peptídeos/genética , Proteínas Quinases/genética , Transativadores/genética , Proteínas rab5 de Ligação ao GTP/genética , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Molecular analysis of a Drosophila minichromosome, Dp(1;f)1187, revealed a relationship between position-effect variegation and the copy number reductions of heterochromatic sequences that occur in polytene cells. Heterochromatin adjacent to a defined junction with euchromatin underpolytenized at least 60-fold. Lesser reductions were observed in euchromatic sequences up to 103 kb from the breakpoint. The copy number changes behaved in all respects like the expression of yellow, a gene located within the affected region. Both copy number and yellow expression displayed a cell-by-cell mosaic pattern of reduction, and adding a Y chromosome, a known suppressor of variegation, increased both substantially. We discuss the possibility that changes in replication alter copy number locally and also propose an alternative model of position-effect variegation based on the somatic elimination of heterochromatic sequences.
Assuntos
Mapeamento Cromossômico , DNA/genética , Drosophila/genética , Animais , Deleção Cromossômica , Drosophila/anatomia & histologia , Feminino , Heterocromatina/fisiologia , Heterocromatina/ultraestrutura , Masculino , Mutação , Hibridização de Ácido Nucleico , Mapeamento por Restrição , Glândulas Salivares/fisiologiaRESUMO
In Drosophila melanogaster males, the sex chromosomes pair during meiosis in the centric X heterochromatin and at the base of the short arm of the Y (YS), in the vicinity of the nucleolus organizers. X chromosomes deficient for the pairing region segregate randomly from the Y. In this report we show that a single ribosomal RNA (rRNA) gene stimulates X-Y pairing and disjunction when inserted onto a heterochromatically deficient X chromosome by P element-mediated transformation. We also show that insert-containing X chromosomes pair at the site of insertion, that autosomal rDNA inserts do not affect X-Y pairing or disjunction, and that the strength of an X pairing site is proportional to the dose of ectopic rRNA genes. These results demonstrate that rRNA genes can promote X-Y pairing and disjunction and imply that the nucleolus organizers function as X-Y pairing sites in wild-type Drosophila males.
Assuntos
DNA Ribossômico/genética , Drosophila melanogaster/genética , Genes , Meiose , RNA Ribossômico/genética , Cromossomo X , Cromossomo Y , Animais , Southern Blotting , Cruzamentos Genéticos , Elementos de DNA Transponíveis , Drosophila melanogaster/fisiologia , Feminino , Masculino , Mutação , Hibridização de Ácido Nucleico , Plasmídeos , Transcrição GênicaRESUMO
The demonstration by Zhang and Spradling (1) of efficient P element transposition into heterochromatic regions will aid ongoing studies of heterochromatin structure and function. P element insertions will provide entry points for further molecular analysis of heterochromatin and will allow the isolation of small and large heterochromatic deficiencies. The generation of heterochromatic P insertions also will aid the study of heterochromatic genes. Of the heterochromatic insertions isolated by Zhang and Spradling, five were homozygous lethal, and one of these defined a lethal locus not previously uncovered by heterochromatic deficiencies. P elements have previously been used to mutagenize and clone specific heterochromatic genes (14, 19, 26). New methods, like those described here (1, 32), should allow the efficient identification and molecular isolation of other single-copy heterochromatic genes. Furthermore, since position-effect suppression allowed the recovery of heterochromatic P insertions, it may also allow the recovery of insertions in euchromatic regions previously refractory to P mutagenesis. Studies of position-effect variegation show that genes normally found in heterochromatin require a heterochromatic context for normal expression and that heterochromatin is inhibitory to euchromatic gene expression (16). The physical basis of these related phenomena--chromatin assembly, nuclear positioning, and/or heterochromatin elimination--can be resolved only with a more thorough understanding of heterochromatin structure and functions. Analyzing heterochromatin also will help define the chromosomal components responsible for inheritance processes such as chromosome pairing, sister chromatid adhesion, and centromere function. These efforts will be facilitated by the effective use of P elements combined with other current molecular-genetic approaches.