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Nature Cell Biology Mar 2022Despite the well-established role of nuclear organization in the regulation of gene expression, little is known about the reverse: how transcription shapes the spatial...
Despite the well-established role of nuclear organization in the regulation of gene expression, little is known about the reverse: how transcription shapes the spatial organization of the genome. Owing to the small sizes of most previously studied genes and the limited resolution of microscopy, the structure and spatial arrangement of a single transcribed gene are still poorly understood. Here we study several long highly expressed genes and demonstrate that they form open-ended transcription loops with polymerases moving along the loops and carrying nascent RNAs. Transcription loops can span across micrometres, resembling lampbrush loops and polytene puffs. The extension and shape of transcription loops suggest their intrinsic stiffness, which we attribute to decoration with multiple voluminous nascent ribonucleoproteins. Our data contradict the model of transcription factories and suggest that although microscopically resolvable transcription loops are specific for long highly expressed genes, the mechanisms underlying their formation could represent a general aspect of eukaryotic transcription.
Topics: Chromosomes; Eukaryota; RNA; Ribonucleoproteins; Transcription, Genetic
PubMed: 35177821
DOI: 10.1038/s41556-022-00847-6 -
Heredity Jul 2019
Review
Topics: Animals; Caenorhabditis elegans; Drosophila; Halobacterium; Models, Genetic; Phycomyces; Polytene Chromosomes; RNA Interference; T-Phages; Tetrahymena
PubMed: 31189909
DOI: 10.1038/s41437-019-0191-5 -
Proceedings of the National Academy of... Jun 2022Cryoelectron tomography of the cell nucleus using scanning transmission electron microscopy and deconvolution processing technology has highlighted a large-scale, 100-...
Cryoelectron tomography of the cell nucleus using scanning transmission electron microscopy and deconvolution processing technology has highlighted a large-scale, 100- to 300-nm interphase chromosome structure, which is present throughout the nucleus. This study further documents and analyzes these chromosome structures. The paper is divided into four parts: 1) evidence (preliminary) for a unified interphase chromosome structure; 2) a proposed unified interphase chromosome architecture; 3) organization as chromosome territories (e.g., fitting the 46 human chromosomes into a 10-μm-diameter nucleus); and 4) structure unification into a polytene chromosome architecture and lampbrush chromosomes. Finally, the paper concludes with a living light microscopy cell study showing that the G1 nucleus contains very similar structures throughout. The main finding is that this chromosome structure appears to coil the 11-nm nucleosome fiber into a defined hollow structure, analogous to a Slinky helical spring [https://en.wikipedia.org/wiki/Slinky; motif used in Bowerman , 10, e65587 (2021)]. This Slinky architecture can be used to build chromosome territories, extended to the polytene chromosome structure, as well as to the structure of lampbrush chromosomes.
Topics: Cell Nucleus; Chromatin; Chromosomes, Human; Humans; Interphase; Nucleosomes
PubMed: 35749363
DOI: 10.1073/pnas.2119101119 -
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 -
Genes Nov 2019In the ciliate somatic macronuclei differentiate from germline micronuclei during sexual reproduction, accompanied by developmental sequence reduction. Concomitantly,... (Review)
Review
In the ciliate somatic macronuclei differentiate from germline micronuclei during sexual reproduction, accompanied by developmental sequence reduction. Concomitantly, over 95% of micronuclear sequences adopt a heterochromatin structure characterized by the histone variant H3.4 and H3K27me3. RNAi-related genes and histone variants dominate the list of developmentally expressed genes. Simultaneously, 27nt-ncRNAs that match sequences retained in new macronuclei are synthesized and bound by PIWI1. Recently, we proposed a mechanistic model for 'RNA-induced DNA replication interference' (RIRI): during polytene chromosome formation PIWI1/27nt-RNA-complexes target macronucleus-destined sequences (MDS) by base-pairing and temporarily cause locally stalled replication. At polytene chromosomal segments with ongoing replication, H3.4K27me3-nucleosomes become selectively deposited, thus dictating the prospective heterochromatin structure of these areas. Consequently, these micronucleus-specific sequences become degraded, whereas 27nt-RNA-covered sites remain protected. However, the biogenesis of the 27nt-RNAs remains unclear. It was proposed earlier that in stichotrichous ciliates 27nt-RNA precursors could derive from telomere-primed bidirectional transcription of nanochromosomes and subsequent Dicer-like (DCL) activity. As a minimalistic explanation, we propose here that the 27nt-RNA precursor could rather be mRNA or pre-mRNA and that the transition of coding RNA from parental macronuclei to non-coding RNAs, which act in premature developing macronuclei, could involve RNA-dependent RNA polymerase (RDRP) activity creating dsRNA intermediates prior to a DCL-dependent pathway. Interestingly, by such mechanism the partition of a parental somatic genome and possibly also the specific nanochromosome copy numbers could be vertically transmitted to the differentiating nuclei of the offspring.
Topics: Ciliophora; DNA Replication; Gene Expression Regulation, Developmental; Genome, Protozoan; Histones; Micronucleus, Germline; Nucleosomes; RNA Interference; RNA Precursors; RNA, Messenger; RNA, Small Nuclear; Telomere
PubMed: 31752243
DOI: 10.3390/genes10110940 -
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 -
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 -
Malaria Journal Mar 2024Anopheles vagus (subgenus Cellia) has been identified as a vector for malaria, filariasis, and Japanese encephalitis in Asia. Sporozoites of Plasmodium falciparum and... (Review)
Review
BACKGROUND
Anopheles vagus (subgenus Cellia) has been identified as a vector for malaria, filariasis, and Japanese encephalitis in Asia. Sporozoites of Plasmodium falciparum and Plasmodium vivax have been found in this zoophilic mosquito in Asia and Indonesia. This study systematically reviews publications regarding An. vagus species, variation, bio-ecology, and malaria transmission in various localities in Asia, especially Indonesia, to determine whether the current data support An. vagus as a species complex.
METHODS
The databases Pubmed, Scopus, Europe PMC, and Proquest were searched to identify information regarding the morphology, karyotypes, polytene chromosome, cross-mating, ecology, and molecular identification of An. vagus was then evaluated to determine whether there were possible species complexes.
RESULTS
Of the 1326 articles identified, 15 studies were considered for synthesis. The Anopheles spp. samples for this study came from Asia. Eleven studies used morphology to identify An. vagus, with singular studies using each of karyotype identification, chromosomal polytene identification, and cross-breeding experiments. Ten studies used molecular techniques to identify Anopheles spp., including An. vagus. Most studies discovered morphological variations of An. vagus either in the same or different areas and ecological settings. In this review, the members of An. vagus sensu lato grouped based on morphology (An. vagus, An. vagus vagus, An. vagus limosus, and An. limosus), karyotyping (form A and B), and molecular (An. vagus genotype A and B, An. vagus AN4 and AN5). Genetic analysis revealed a high conservation of the ITS2 fragment among members except for the An. vagus genotype B, which was, in fact, Anopheles sundaicus. This review also identified that An. vagus limosus and An. vagus vagus were nearly identical to the ITS2 sequence.
CONCLUSION
Literature review studies revealed that An. vagus is conspecific despite the distinct morphological characteristic of An. vagus and An. limosus. Further information using another barcoding tool, such as mitochondrial COI and ND6 and experimental cross-mating between the An. vagus and An. limosus may provide additional evidence for the status of An. vagus as a species complex.
Topics: Animals; Phylogeny; Anopheles; Genotype; Mosquito Vectors; Malaria
PubMed: 38539155
DOI: 10.1186/s12936-024-04888-0 -
Insects Feb 2021The genome assembly of consists of 2221 scaffolds (N50 = 115,072 bp) and has a size spanning 136.94 Mbp. This assembly represents one of the smallest genomes among...
The genome assembly of consists of 2221 scaffolds (N50 = 115,072 bp) and has a size spanning 136.94 Mbp. This assembly represents one of the smallest genomes among species. genomic DNA fragments of ~37 Kb were cloned, end-sequenced, and used as probes for fluorescence in situ hybridization (FISH) with salivary gland polytene chromosomes. In total, we mapped nine DNA probes to scaffolds and autosomal arms. Comparative analysis of the scaffolds with homologous sequences of the and genomes identified chromosomal rearrangements among these species. Our results confirmed that physical mapping is a useful tool for anchoring genome assemblies to mosquito chromosomes.
PubMed: 33671870
DOI: 10.3390/insects12020164