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Cells Nov 2020Dipterans exhibit a remarkable diversity of chromosome end structures in contrast to the conserved system defined by telomerase and short repeats. Within dipteran...
BACKGROUND
Dipterans exhibit a remarkable diversity of chromosome end structures in contrast to the conserved system defined by telomerase and short repeats. Within dipteran families, structure of chromosome termini is usually conserved within genera. With the aim to assess whether or not the evolutionary distance between genera implies chromosome end diversification, this report exploits two representatives of Sciaridae, , and .
METHODS
Probes and plasmid microlibraries obtained by chromosome end microdissection, in situ hybridization, cloning, and sequencing are among the methodological approaches employed in this work.
RESULTS
The data argue for the existence of either specific terminal DNA sequences for each chromosome tip in , or sequences common to all chromosome ends but their extension does not allow detection by in situ hybridization. Both sciarid species share terminal sequences that are significantly underrepresented in chromosome ends of .
CONCLUSIONS
The data suggest an unusual terminal structure in chromosomes compared to other dipterans investigated. A putative, evolutionary process of repetitive DNA expansion that acted differentially to shape chromosome ends of the two flies is also discussed.
Topics: Animals; Base Sequence; Chromosomes, Insect; DNA; Diptera; Gene Library; Microdissection; Plasmids; Polytene Chromosomes
PubMed: 33167604
DOI: 10.3390/cells9112425 -
Insects Apr 2022The represent a diverse group of closely related to Although they have radiated extensively in Australia, they have been the focus of few studies. Here, we...
The represent a diverse group of closely related to Although they have radiated extensively in Australia, they have been the focus of few studies. Here, we characterized the karyotypes of 12 species from several species groups and showed that they have undergone similar types of karyotypic change to those seen in . This includes heterochromatin amplification involved in length changes of the sex and 'dot' chromosomes as well as the autosomes, particularly in the group of species. Numerous weak points along the arms of the polytene chromosomes suggest the presence of internal repetitive sequence DNA, but these regions did not C-band in mitotic chromosomes, and their analysis will depend on DNA sequencing. The nucleolar organizing regions (NORs) are at the same chromosome positions in as in and the various mechanisms responsible for changing arm configurations also appear to be the same. These chromosomal studies provide a complementary resource to other investigations of this group, with several species currently being sequenced.
PubMed: 35447805
DOI: 10.3390/insects13040364 -
Current Genomics Apr 2018This mini-review is devoted to the problem genetic meaning of main polytene chromosome structures - bands and interbands. Generally, densely packed chromatin forms black... (Review)
Review
This mini-review is devoted to the problem genetic meaning of main polytene chromosome structures - bands and interbands. Generally, densely packed chromatin forms black bands, moderately condensed regions form grey loose bands, whereas decondensed regions of the genome appear as interbands. Recent progress in the annotation of the Drosophila genome and epigenome has made it possible to compare the banding pattern and the structural organization of genes, as well as their activity. This was greatly aided by our ability to establish the borders of bands and interbands on the physical map, which allowed to perform comprehensive side-by-side comparisons of cytology, genetic and epigenetic maps and to uncover the association between the morphological structures and the functional domains of the genome. These studies largely conclude that interbands 5'-ends of housekeeping genes that are active across all cell types. Interbands are enriched with proteins involved in transcription and nucleosome remodeling, as well as with active histone modifications. Notably, most of the replication origins map to interband regions. As for grey loose bands adjacent to interbands, they typically host the bodies of house-keeping genes. Thus, the bipartite structure composed of an interband and an adjacent grey band functions as a standalone genetic unit. Finally, black bands harbor tissue-specific genes with narrow temporal and tissue expression profiles. Thus, the uniform and permanent activity of interbands combined with the inactivity of genes in bands forms the basis of the universal banding pattern observed in various Drosophila tissues.
PubMed: 29606905
DOI: 10.2174/1389202918666171016123830 -
Cells Feb 2020Spatial organization of chromosome territories and interactions between interphase chromosomes themselves, as well as with the nuclear periphery, play important roles in...
Spatial organization of chromosome territories and interactions between interphase chromosomes themselves, as well as with the nuclear periphery, play important roles in epigenetic regulation of the genome function. However, the interplay between inter-chromosomal contacts and chromosome-nuclear envelope attachments in an organism's development is not well-understood. To address this question, we conducted microscopic analyses of the three-dimensional chromosome organization in malaria mosquitoes. We employed multi-colored oligonucleotide painting probes, spaced 1 Mb apart along the euchromatin, to quantitatively study chromosome territories in larval salivary gland cells and adult ovarian nurse cells of , , and . We found that the X chromosome territory has a significantly smaller volume and is more compact than the autosomal arm territories. The number of inter-chromosomal, and the percentage of the chromosome-nuclear envelope, contacts were conserved among the species within the same cell type. However, the percentage of chromosome regions located at the nuclear periphery was typically higher, while the number of inter-chromosomal contacts was lower, in salivary gland cells than in ovarian nurse cells. The inverse correlation was considerably stronger for the autosomes. Consistent with previous theoretical arguments, our data indicate that, at the genome-wide level, there is an inverse relationship between chromosome-nuclear envelope attachments and chromosome-chromosome interactions, which is a key feature of the cell type-specific nuclear architecture.
Topics: Animals; Anopheles; Female; Germ Cells; Malaria; Nuclear Envelope; Ovary; Polytene Chromosomes; Salivary Glands; X Chromosome
PubMed: 32024176
DOI: 10.3390/cells9020339 -
PloS One 2012In D. melanogaster polytene chromosomes, intercalary heterochromatin (IH) appears as large dense bands scattered in euchromatin and comprises clusters of repressed...
In D. melanogaster polytene chromosomes, intercalary heterochromatin (IH) appears as large dense bands scattered in euchromatin and comprises clusters of repressed genes. IH displays distinctly low gene density, indicative of their particular regulation. Genes embedded in IH replicate late in the S phase and become underreplicated. We asked whether localization and organization of these late-replicating domains is conserved in a distinct cell type. Using published comprehensive genome-wide chromatin annotation datasets (modENCODE and others), we compared IH organization in salivary gland cells and in a Kc cell line. We first established the borders of 60 IH regions on a molecular map, these regions containing underreplicated material and encompassing ∼12% of Drosophila genome. We showed that in Kc cells repressed chromatin constituted 97% of the sequences that corresponded to IH bands. This chromatin is depleted for ORC-2 binding and largely replicates late. Differences in replication timing between the cell types analyzed are local and affect only sub-regions but never whole IH bands. As a rule such differentially replicating sub-regions display open chromatin organization, which apparently results from cell-type specific gene expression of underlying genes. We conclude that repressed chromatin organization of IH is generally conserved in polytene and non-polytene cells. Yet, IH domains do not function as transcription- and replication-regulatory units, because differences in transcription and replication between cell types are not domain-wide, rather they are restricted to small "islands" embedded in these domains. IH regions can thus be defined as a special class of domains with low gene density, which have narrow temporal expression patterns, and so displaying relatively conserved organization.
Topics: Animals; Base Sequence; DNA Replication; DNA Replication Timing; Drosophila melanogaster; Heterochromatin; Insect Proteins; Physical Chromosome Mapping; Polytene Chromosomes
PubMed: 22253867
DOI: 10.1371/journal.pone.0030035 -
Journal of Visualized Experiments : JoVE Jan 2014Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal...
Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.
Topics: Animals; Anopheles; Chromosome Painting; DNA; Female; In Situ Hybridization, Fluorescence; Insect Vectors; Laser Capture Microdissection
PubMed: 24429496
DOI: 10.3791/51173 -
Genes Feb 2020underreplicate the DNA of thoracic nuclei, stalling during S phase at a point that is proportional to the total genome size in each species. In polytene tissues, such...
underreplicate the DNA of thoracic nuclei, stalling during S phase at a point that is proportional to the total genome size in each species. In polytene tissues, such as the salivary glands, all of the nuclei initiate multiple rounds of DNA synthesis and underreplicate. Yet, only half of the nuclei isolated from the thorax stall; the other half do not initiate S phase. Our question was, why half? To address this question, we use flow cytometry to compare underreplication phenotypes between thoracic tissues. When individual thoracic tissues are dissected and the proportion of stalled DNA synthesis is scored in each tissue type, we find that underreplication occurs in the indirect flight muscle, with the majority of underreplicated nuclei in the dorsal longitudinal muscles (DLM). Half of the DNA in the DLM nuclei stall at S phase between the unreplicated G0 and fully replicated G1. The dorsal ventral flight muscle provides the other source of underreplication, and yet, there, the replication stall point is earlier (less DNA replicated), and the endocycle is initiated. The differences in underreplication and ploidy in the indirect flight muscles provide a new tool to study heterochromatin, underreplication and endocycle control.
Topics: Animals; Cell Nucleus; DNA; DNA Replication; Drosophila melanogaster; Flight, Animal; Flow Cytometry; G1 Phase; Muscle, Skeletal; Polytene Chromosomes; Resting Phase, Cell Cycle; S Phase; Salivary Glands; Thorax
PubMed: 32111003
DOI: 10.3390/genes11030246 -
Experimental Biology and Medicine... Nov 2019Repairing DNA double-strand breaks is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of...
UNLABELLED
Repairing DNA double-strand breaks is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination. In Kc cells, accurate homologous recombination repair of heterochromatic double-strand breaks relies on the relocalization of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. This movement is driven by Arp2/3-dependent nuclear actin filaments and myosins’ ability to walk along them. Conserved mechanisms enable the relocalization of heterochromatic repair sites in mouse cells, and defects in these pathways lead to massive ectopic recombination in heterochromatin and chromosome rearrangements. In polytene chromosomes, extensive DNA movement is blocked by a stiff structure of chromosome bundles. Repair pathways in this context are poorly characterized, and whether heterochromatic double-strand breaks relocalize in these cells is unknown. Here, we show that damage in heterochromatin results in relaxation of the heterochromatic chromocenter, consistent with a dynamic response. Arp2/3, the Arp2/3 activator Scar, and the myosin activator Unc45, are required for heterochromatin stability in polytene cells, suggesting that relocalization enables heterochromatin repair also in this tissue. Together, these studies reveal critical roles for actin polymerization and myosin motors in heterochromatin repair and genome stability across different organisms and tissue types.
IMPACT STATEMENT
Heterochromatin relies on dedicated pathways for ‘safe’ recombinational repair. In mouse and fly cultured cells, DNA double-strand break repair requires the movement of damaged sites away from the heterochromatin ‘domain’ nuclear actin filaments and myosins. Here, we explore the importance of these pathways in salivary gland cells, which feature a stiff bundle of endoreduplicated polytene chromosomes. Repair pathways in polytene chromosomes are largely obscure and how nuclear dynamics operate in this context is unknown. We show that heterochromatin relaxes in response to damage, and relocalization pathways are necessary to prevent abnormal repair and promote the stability of heterochromatic sequences. These results deepen our understanding of DNA damage response mechanisms in polytene chromosomes, revealing unexpected dynamics. It also provides a first understanding of nuclear dynamics responding to replication damage and rDNA breaks, providing a new understanding of the importance of nuclear architecture in genome stability. We expect these discoveries will shed light on tumorigenic processes, including therapy-induced cancer relapses.
Topics: Actin Cytoskeleton; Actin-Related Protein 2-3 Complex; Animals; DNA Breaks, Double-Stranded; DNA Repair; Drosophila Proteins; Drosophila melanogaster; Genomic Instability; Heterochromatin; Molecular Chaperones; Myosins; Polytene Chromosomes
PubMed: 31364400
DOI: 10.1177/1535370219862282 -
Cell Cycle (Georgetown, Tex.) Jan 2017
Topics: Amyloid; Animals; Chromosomal Puffs; Mitochondria; Models, Biological; Oocytes; Oogenesis; Xenopus; Xenopus Proteins
PubMed: 27736303
DOI: 10.1080/15384101.2016.1241605 -
Biochemistry. Biokhimiia Apr 2018Drosophila polytene chromosomes are widely used as a model of eukaryotic interphase chromosomes. The most noticeable feature of polytene chromosome is transverse banding... (Review)
Review
Drosophila polytene chromosomes are widely used as a model of eukaryotic interphase chromosomes. The most noticeable feature of polytene chromosome is transverse banding associated with alternation of dense stripes (dark or black bands) and light diffuse areas that encompass alternating less compact gray bands and interbands visible with an electron microscope. In recent years, several approaches have been developed to predict location of morphological structures of polytene chromosomes based on the distribution of proteins on the molecular map of Drosophila genome. Comparison of these structures with the results of analysis of the three-dimensional chromatin organization by the Hi-C method indicates that the morphology of polytene chromosomes represents direct visualization of the interphase nucleus spatial organization into topological domains. Compact black bands correspond to the extended topological domains of inactive chromatin, while interbands are the barriers between the adjacent domains. Here, we discuss the prospects of using polytene chromosomes to study mechanisms of spatial organization of interphase chromosomes, as well as their dynamics and evolution.
Topics: Animals; Chromatin; Chromatin Assembly and Disassembly; Drosophila; Polytene Chromosomes
PubMed: 29626921
DOI: 10.1134/S0006297918040053