RESUMO
Karyotypes, composed of chromosomes, must be accurately partitioned by the mitotic spindle for optimal cell health. However, it is unknown how underlying characteristics of karyotypes, such as chromosome number and size, govern the scaling of the mitotic spindle to ensure accurate chromosome segregation and cell proliferation. We utilize budding yeast strains engineered with fewer chromosomes, including just two "mega chromosomes," to study how spindle size and function are responsive to, and scaled by, karyotype. We determined that deletion and overexpression of spindle-related genes are detrimental to the growth of strains with two chromosomes, suggesting that mega chromosomes exert altered demands on the spindle. Using confocal microscopy, we demonstrate that cells with fewer but longer chromosomes have smaller spindle pole bodies, fewer microtubules, and longer spindles. Moreover, using electron tomography and confocal imaging, we observe elongated, bent anaphase spindles with fewer core microtubules in strains with mega chromosomes. Cells harboring mega chromosomes grow more slowly, are delayed in mitosis, and a subset struggle to complete chromosome segregation. We propose that the karyotype of the cell dictates the microtubule number, type, spindle pole body size, and spindle length, subsequently influencing the dynamics of mitosis, such as the rate of spindle elongation and the velocity of pole separation. Taken together, our results suggest that mitotic spindles are highly plastic ultrastructures that can accommodate and adjust to a variety of karyotypes, even within a species.
Assuntos
Saccharomyces cerevisiae , Fuso Acromático , Fuso Acromático/metabolismo , Saccharomyces cerevisiae/genética , Microtúbulos/metabolismo , Segregação de Cromossomos , Mitose , Cromossomos Fúngicos/genética , CariótipoRESUMO
A properly regulated series of developmental and meiotic events must occur to ensure the successful production of gametes. In Drosophila melanogaster ovaries, these early developmental and meiotic events include the production of the 16-cell cyst, meiotic entry, synaptonemal complex (SC) formation, recombination, and oocyte specification. In order to identify additional genes involved in early oocyte development and meiosis, we reanalyzed 3 published single-cell RNA-seq datasets from Drosophila ovaries, using vasa (germline) together with c(3)G, cona, and corolla (SC) as markers. Our analysis generated a list of 2,743 co-expressed genes. Many known SC-related and early oocyte development genes fell within the top 500 genes on this list, as ranked by the abundance and specificity of each gene's expression across individual analyses. We tested 526 available RNAi lines containing shRNA constructs in germline-compatible vectors representing 331 of the top 500 genes. We assessed targeted ovaries for SC formation and maintenance, oocyte specification, cyst development, and double-strand break dynamics. Six uncharacterized genes exhibited early developmental defects. SC and developmental defects were observed for additional genes not well characterized in the early ovary. Interestingly, in some lines with developmental delays, meiotic events could still be completed once oocyte specificity occurred indicating plasticity in meiotic timing. These data indicate that a transcriptomics approach can be used to identify genes involved in functions in a specific cell type in the Drosophila ovary.
Assuntos
Cistos , Proteínas de Drosophila , Animais , Feminino , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Interferência de RNA , Recombinação Genética , Complexo Sinaptonêmico , Meiose/genética , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Oócitos/metabolismo , Perfilação da Expressão Gênica , Cistos/genética , Cistos/metabolismoRESUMO
Although Lepidopteran females build a synaptonemal complex (SC) in pachytene, homologs do not crossover, necessitating an alternative method of homolog conjunction. In Bombyx mori oocytes, the SC breaks down at the end of pachytene, and homolog associations are maintained by a large oocyte-specific structure, which we call the bivalent bridge (BB), connecting paired homologs. The BB is derived from at least some components of the SC lateral elements (LEs). It contains the HORMAD protein HOP1 and the LE protein SYCP2 and is formed by the fusion of the two LE derivatives. As diplotene progresses, the BB increases in width and acquires a layered structure with a thick band of HOP1 separating two layers of SYCP2. The HOP1 interacting protein, PCH2, joins the BB in mid-diplotene, and by late-diplotene, it lies in the middle of the HOP1 filament. This structure is maintained through metaphase I. SYCP2 and PCH2 are lost at anaphase I, and the BB no longer connects the separating homologs. However, a key component of the BB, HOP1, remains at the metaphase I plate. These changes in organization of the BB occur simultaneously with the movement of the kinetochore protein, DSN1, from within the BB at mid-diplotene to the edge of the homologs facing the poles by metaphase I. We view these data in context of models in which SC components and regulators can be repurposed to achieve different functions, a fascinating example of evolution achieving homolog conjunction in an alternative way with recycling of SC proteins.
Assuntos
Bombyx , Complexo Sinaptonêmico , Animais , Feminino , Meiose , Oócitos/metabolismo , MetáfaseRESUMO
In almost all sexually reproducing organisms, meiotic recombination and cell division require the synapsis of homologous chromosomes by a large proteinaceous structure, the synaptonemal complex (SC). While the SC's overall structure is highly conserved across eukaryotes, its constituent proteins diverge between phyla. Transverse filament protein, SYCP1, spans the width of the SC and undergoes amino-terminal head-to-head self-assembly in vitro through a motif that is unusually highly conserved across kingdoms of life. Here, we report creation of mouse mutants, Sycp1L102E and Sycp1L106E, that target SYCP1's head-to-head interface. L106E resulted in a complete loss of synapsis, while L102E had no apparent effect on synapsis, in agreement with their differential effects on the SYCP1 head-to-head interface in molecular dynamics simulations. In Sycp1L106E mice, homologs aligned and recruited low levels of mutant SYCP1 and other SC proteins, but the absence of synapsis led to failure of crossover formation and meiotic arrest. We conclude that SYCP1's conserved head-to-head interface is essential for meiotic chromosome synapsis in vivo.
Assuntos
Pareamento Cromossômico , Proteínas Nucleares , Animais , Camundongos , Recombinação Homóloga , Meiose/genética , Proteínas Nucleares/metabolismo , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismoRESUMO
Selfish genetic elements use a myriad of mechanisms to drive their inheritance and ensure their survival into the next generation, often at a fitness cost to its host.1,2 Although the catalog of selfish genetic elements is rapidly growing, our understanding of host drive suppression systems that counteract self-seeking behavior is lacking. Here, we demonstrate that the biased transmission of the non-essential, non-driving B chromosomes in Drosophila melanogaster can be achieved in a specific genetic background. Combining a null mutant of matrimony, a gene that encodes a female-specific meiotic regulator of Polo kinase,3,4 with the TM3 balancer chromosome creates a driving genotype that is permissive for the biased transmission of the B chromosomes. This drive is female-specific, and both genetic components are necessary, but not individually sufficient, for permitting a strong drive of the B chromosomes. Examination of metaphase I oocytes reveals that B chromosome localization within the DNA mass is mostly abnormal when drive is the strongest, indicating a failure of the mechanism(s) responsible for the proper distribution of B chromosomes. We propose that some proteins important for proper chromosome segregation during meiosis, like Matrimony, may have an essential role as part of a meiotic drive suppression system that modulates chromosome segregation to prevent genetic elements from exploiting the inherent asymmetry of female meiosis.
Assuntos
Drosophila melanogaster , Padrões de Herança , Animais , Feminino , Drosophila melanogaster/genética , Meiose/genética , Cromossomos/genéticaRESUMO
Meiosis is usually described as 4 essential and sequential processes: (1) homolog pairing; (2) synapsis, mediated by the synaptonemal complex; (3) crossing over; and (4) segregation. In this canonical model, the maturation of crossovers into chiasmata plays a vital role in holding homologs together and ensuring their segregation at the first meiotic division. However, Lepidoptera (moths and butterflies) undergo 3 distinct meiotic processes, only one of which is canonical. Lepidoptera males utilize 2 meiotic processes: canonical meiosis that produces nucleated fertile sperm, and a noncanonical meiosis that produces anucleated nonfertile sperm which are nonetheless essential for reproduction. Lepidoptera females, which carry heteromorphic sex chromosomes, undergo a completely achiasmate (lacking crossovers) meiosis, thereby requiring an alternative mechanism to ensure proper homolog segregation. Here, we report that the development of a molecular cell biology toolkit designed to properly analyze features of meiosis, including the synaptonemal complex structure and function, in the silkworm Bombyx mori. In addition to standard homology searches to identify Bombyx orthologs of known synaptonemal complex encoding genes, we developed an ortholog discovery app (Shinyapp) to identify Bombyx orthologs of proteins involved in several meiotic processes. We used this information to clone genes expressed in the testes and then created antibodies against their protein products. We used the antibodies to confirm the localization of these proteins in normal male spermatocytes, as well as using in vitro assays to confirm orthologous interactions. The development of this toolkit will facilitate further study of the unique meiotic processes that characterize meiosis in Lepidoptera.
Assuntos
Bombyx , Borboletas , Animais , Feminino , Masculino , Bombyx/genética , Borboletas/genética , Sêmen , Pareamento Cromossômico , Complexo Sinaptonêmico , Cromossomos Sexuais , MeioseRESUMO
Transposable elements (TE) are selfish genetic elements that can cause harmful mutations. In Drosophila, it has been estimated that half of all spontaneous visible marker phenotypes are mutations caused by TE insertions. Several factors likely limit the accumulation of exponentially amplifying TEs within genomes. First, synergistic interactions between TEs that amplify their harm with increasing copy number are proposed to limit TE copy number. However, the nature of this synergy is poorly understood. Second, because of the harm posed by TEs, eukaryotes have evolved systems of small RNA-based genome defense to limit transposition. However, as in all immune systems, there is a cost of autoimmunity and small RNA-based systems that silence TEs can inadvertently silence genes flanking TE insertions. In a screen for essential meiotic genes in Drosophila melanogaster, a truncated Doc retrotransposon within a neighboring gene was found to trigger the germline silencing of ald, the Drosophila Mps1 homolog, a gene essential for proper chromosome segregation in meiosis. A subsequent screen for suppressors of this silencing identified a new insertion of a Hobo DNA transposon in the same neighboring gene. Here we describe how the original Doc insertion triggers flanking piRNA biogenesis and local gene silencing. We show that this local gene silencing occurs in cis and is dependent on deadlock, a component of the Rhino-Deadlock-Cutoff (RDC) complex, to trigger dual-strand piRNA biogenesis at TE insertions. We further show how the additional Hobo insertion leads to de-silencing by reducing flanking piRNA biogenesis triggered by the original Doc insertion. These results support a model of TE-mediated gene silencing by piRNA biogenesis in cis that depends on local determinants of transcription. This may explain complex patterns of off-target gene silencing triggered by TEs within populations and in the laboratory. It also provides a mechanism of sign epistasis among TE insertions, illuminates the complex nature of their interactions and supports a model in which off-target gene silencing shapes the evolution of the RDC complex.
Assuntos
Drosophila melanogaster , RNA de Interação com Piwi , Animais , Drosophila melanogaster/genética , Elementos de DNA Transponíveis , RNA Interferente Pequeno/genética , Drosophila/genética , Inativação GênicaRESUMO
The rapidly evolving stem cell field puts much stress on developing educational resources. The ISSCR Education Committee has created a flexible stem cell syllabus rooted in core concepts to facilitate stem cell literacy. The free syllabus will be updated regularly to maintain accuracy and relevance.
Assuntos
Currículo , Alfabetização , Células-TroncoRESUMO
First discovered in maize, paramutation is a phenomenon in which one allele can trigger an epigenetic conversion of an alternate allele. This conversion causes a genetically heterozygous individual to transmit alleles that are functionally the same, in apparent violation of Mendelian segregation. Studies over the past several decades have revealed a strong connection between mechanisms of genome defense against transposable elements by small RNA and the phenomenon of paramutation. For example, a system of paramutation in Drosophila melanogaster has been shown to be mediated by piRNAs, whose primary function is to silence transposable elements in the germline. In this paper, we characterize a second system of piRNA-mediated paramutation-like behavior at the telomere of Drosophila virilis. In Drosophila, telomeres are maintained by arrays of retrotransposons that are regulated by piRNAs. As a result, the telomere and sub-telomeric regions of the chromosome have unique regulatory and chromatin properties. Previous studies have shown that maternally deposited piRNAs derived from a sub-telomeric piRNA cluster can silence the sub-telomeric center divider gene of Drosophila virilis in trans. In this paper, we show that this silencing can also be maintained in the absence of the original silencing allele in a subsequent generation. The precise mechanism of this paramutation-like behavior may be explained by either the production of retrotransposon piRNAs that differ across strains or structural differences in the telomere. Altogether, these results show that the capacity for piRNAs to mediate paramutation in trans may depend on the local chromatin environment and proximity to the uniquely structured telomere regulated by piRNAs. This system promises to provide significant insights into the mechanisms of paramutation.
RESUMO
Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. We show that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species.
Assuntos
Drosophila melanogaster/genética , Tamanho do Genoma , Genômica/métodos , Animais , Linhagem Celular , Cromossomos , Biologia Computacional/métodos , Feminino , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , NanoporosRESUMO
Cathleen Lake and Scott Hawley discuss the components, assembly and functional importance of the synaptonemal complex.
Assuntos
Troca Genética , Meiose , Complexo Sinaptonêmico , Animais , Pareamento Cromossômico , Segregação de Cromossomos , Humanos , Meiose/genética , Schizosaccharomyces/citologia , Complexo Sinaptonêmico/química , Complexo Sinaptonêmico/metabolismoRESUMO
Experiments performed in different genetic backgrounds occasionally exhibit failure in experimental reproducibility. This is a serious issue in Drosophila where there are no standard control stocks. Here, we illustrate the importance of controlling genetic background by showing that the timing of a major meiotic event, the breakdown of the synaptonemal complex (SC), varies in different genetic backgrounds. We assessed SC breakdown in three different control stocks and found that in one control stock, y w; svspa-pol, the SC broke down earlier than in Oregon-R and w1118 stocks. We further examined SC breakdown in these three control backgrounds with flies heterozygous for a null mutation in c(3)G, which encodes a key structural component of the SC. Flies heterozygous for c(3)G displayed differences in the timing of SC breakdown in different control backgrounds, providing evidence of a sensitizing effect of this mutation. These observations suggest that SC maintenance is associated with the dosage of c(3)G in some backgrounds. Lastly, chromosome segregation was not affected by premature SC breakdown in mid-prophase, consistent with previous findings that chromosome segregation is not dependent on full-length SC in mid-prophase. Thus, genetic background is an important variable to consider with respect to SC behavior during Drosophila meiosis.
Assuntos
Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Patrimônio Genético , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismo , Alelos , Animais , Diferenciação Celular , Segregação de Cromossomos , Drosophila melanogaster/citologia , Desenvolvimento Embrionário , Estudos de Associação Genética , Genótipo , Heterozigoto , Imagem Molecular , Mutação , Oócitos/citologia , Oócitos/metabolismoRESUMO
Balancers are rearranged chromosomes used in Drosophila melanogaster to maintain deleterious mutations in stable populations, preserve sets of linked genetic elements and construct complex experimental stocks. Here, we assess the phenotypes associated with breakpoint-induced mutations on commonly used third chromosome balancers and show remarkably few deleterious effects. We demonstrate that a breakpoint in p53 causes loss of radiation-induced apoptosis and a breakpoint in Fucosyltransferase A causes loss of fucosylation in nervous and intestinal tissue-the latter study providing new markers for intestinal cell identity and challenging previous conclusions about the regulation of fucosylation. We also describe thousands of potentially harmful mutations shared among X or third chromosome balancers, or unique to specific balancers, including an Ankyrin2 mutation present on most TM3 balancers, and reiterate the risks of using balancers as experimental controls. We used long-read sequencing to confirm or refine the positions of two inversions with breakpoints lying in repetitive sequences and provide evidence that one of the inversions, In(2L)Cy, arose by ectopic recombination between foldback transposon insertions and the other, In(3R)C, cleanly separates subtelomeric and telomeric sequences and moves the subtelomeric sequences to an internal chromosome position. In addition, our characterization of In(3R)C shows that balancers may be polymorphic for terminal deletions. Finally, we present evidence that extremely distal mutations on balancers can add to the stability of stocks whose purpose is to maintain homologous chromosomes carrying mutations in distal genes. Overall, these studies add to our understanding of the structure, diversity and effectiveness of balancer chromosomes.
Assuntos
Cromossomos , Drosophila melanogaster , Animais , Inversão Cromossômica , Drosophila melanogaster/genética , Mutação , FenótipoRESUMO
The synaptonemal complex (SC), a highly conserved structure built between homologous meiotic chromosomes, is required for crossover formation and ensuring proper chromosome segregation. In many organisms, SC components can also form alternative structures, including repeating SC structures that are known as polycomplexes (PCs), and extensively modified SC structures that are maintained late in meiosis. PCs display differences in their ability to localize with lateral element proteins, recombination machinery, and DNA. They can be created by defects in post-translational modification, suggesting that these modifications have roles in preventing alternate SC structures. These SC-like structures provide insight into the rules for building and maintaining the SC by offering an 'in vivo laboratory' for models of SC assembly, structure, and disassembly. Here, we discuss what these structures can tell us about the rules for building the SC and the roles of the SC in meiotic processes.
Assuntos
Pareamento Cromossômico , Segregação de Cromossomos , Troca Genética , Meiose , Proteínas Nucleares/genética , Complexo Sinaptonêmico , Animais , HumanosRESUMO
The proper behavior of homologous chromosomes at the first meiotic division is usually ensured by crossing over. A new study shows that crossover position influences the successful completion of the chromatin remodeling processes that facilitate homologous segregation.
Assuntos
Segregação de Cromossomos , Troca Genética , Cromossomos/genética , MeioseRESUMO
Polo-like kinases (PLKs) have numerous roles in both mitosis and meiosis, including functions related to chromosome segregation, cohesin removal, and kinetochore orientation [1-7]. PLKs require specific regulation during meiosis to control those processes. Genetic studies demonstrate that the Drosophila PLK Polo kinase (Polo) is inhibited by the female meiosis-specific protein Matrimony (Mtrm) in a stoichiometric manner [8]. Drosophila Polo localizes strongly to kinetochores and to central spindle microtubules during prometaphase and metaphase I of female meiosis [9, 10]. Mtrm protein levels increase dramatically after nuclear envelope breakdown [11]. We show that Mtrm is enriched along the meiotic spindle and that loss of mtrm results in mislocalization of the catalytically active form of Polo. The mtrm gene is haploinsufficient, and heterozygosity for mtrm (mtrm/+) results in high levels of achiasmate chromosome missegregation [8, 12]. In mtrm/+ heterozygotes, there is a low level of sister centromere separation, as well as precocious loss of cohesion along the arms of achiasmate chromosomes. However, mtrm-null females are sterile [13], and sister chromatid cohesion is abolished on all chromosomes, leading to a failure to properly congress or orient chromosomes in metaphase I. These data demonstrate a requirement for the inhibition of Polo, perhaps by sequestering Polo to the microtubules during Drosophila melanogaster female meiosis and suggest that catalytically active Polo is a distinct subset of the total Polo population within the oocyte that requires its own regulation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Meiose , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Feminino , CoesinasRESUMO
Balancer chromosomes are multiply inverted and rearranged chromosomes that are widely used in Drosophila genetics. First described nearly 100 years ago, balancers are used extensively in stock maintenance and complex crosses. Recently, the complete molecular structures of several commonly used balancers were determined by whole-genome sequencing. This revealed a surprising amount of variation among balancers derived from a common progenitor, identified genes directly affected by inversion breakpoints, and cataloged mutations shared by balancers. These studies emphasized that it is important to choose the optimal balancer, because different inversions suppress meiotic recombination in different chromosomal regions. In this review, we provide a brief history of balancers in Drosophila, discuss how they are used today, and provide examples of unexpected recombination events involving balancers that can lead to stock breakdown.
Assuntos
Inversão Cromossômica/genética , Cromossomos de Insetos/genética , Heterocromatina/genética , Recombinação Homóloga/genética , Animais , Centrômero/genética , Drosophila melanogaster/genética , Genoma de Inseto/genética , Fenótipo , Deleção de Sequência/genética , Cromossomo X/genéticaRESUMO
The synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double-strand breaks (DSBs) into crossovers or gene conversions. The removal of any central-region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, we used CRISPR/Cas9 to construct 3 in-frame deletions within the predicted coiled-coil region of the C(3)G protein. Since these 3 deletion mutations disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, they allow us to define the stages of pachytene when the SC is necessary for homolog pairing and recombination during pachytene. Our studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently from the autosomes.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Estágio Paquíteno/genética , Complexo Sinaptonêmico/genética , Cromossomo X/genética , Animais , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Recombinação Genética/genética , Deleção de Sequência/genéticaRESUMO
During early meiotic prophase, homologous chromosomes are connected along their entire lengths by a proteinaceous tripartite structure known as the synaptonemal complex (SC). Although the components that comprise the SC are predominantly studied in this canonical ribbon-like structure, they can also polymerize into repeated structures known as polycomplexes. We find that in Drosophila oocytes, the ability of SC components to assemble into canonical tripartite SC requires the E3 ubiquitin ligase Seven in absentia (Sina). In sina mutant oocytes, SC components assemble into large rod-like polycomplexes instead of proper SC. Thus, the wild-type Sina protein inhibits the polymerization of SC components, including those of the lateral element, into polycomplexes. These polycomplexes persist into meiotic stages when canonical SC has been disassembled, indicating that Sina also plays a role in controlling SC disassembly. Polycomplexes induced by loss-of-function sina mutations associate with centromeres, sites of double-strand breaks, and cohesins. Perhaps as a consequence of these associations, centromere clustering is defective and crossing over is reduced. These results suggest that while features of the polycomplexes can be recognized as SC by other components of the meiotic nucleus, polycomplexes nonetheless fail to execute core functions of canonical SC.