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Genome Research Mar 2015Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial...
Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.
Topics: Animals; Chromosome Mapping; Chromosomes, Artificial, Bacterial; Computational Biology; Contig Mapping; Drosophila melanogaster; Genome; High-Throughput Nucleotide Sequencing; In Situ Hybridization, Fluorescence; Molecular Sequence Data; Polytene Chromosomes; Restriction Mapping
PubMed: 25589440
DOI: 10.1101/gr.185579.114 -
Insects Sep 2018Anopheline mosquitoes are important vectors of human malaria. Next-generation sequencing opens new opportunities for studies of mosquito genomes to uncover the genetic...
Anopheline mosquitoes are important vectors of human malaria. Next-generation sequencing opens new opportunities for studies of mosquito genomes to uncover the genetic basis of a transmission. Physical mapping of genome sequences to polytene chromosomes significantly improves reference assemblies. High-resolution cytogenetic maps are essential for anchoring genome sequences to chromosomes as well as for studying breakpoints of chromosome rearrangements and chromatin protein localization. Here we describe a detailed pipeline for the development of high-resolution cytogenetic maps using polytene chromosomes of malaria mosquitoes. We apply this workflow to the refinement of the cytogenetic map developed for
PubMed: 30227611
DOI: 10.3390/insects9030121 -
Nucleic Acids Research Jan 2016Increasing amounts of data support a role for guanine quadruplex (G4) DNA and RNA structures in various cellular processes. We stained different organisms with...
Increasing amounts of data support a role for guanine quadruplex (G4) DNA and RNA structures in various cellular processes. We stained different organisms with monoclonal antibody 1H6 specific for G4 DNA. Strikingly, immuno-electron microscopy showed exquisite specificity for heterochromatin. Polytene chromosomes from Drosophila salivary glands showed bands that co-localized with heterochromatin proteins HP1 and the SNF2 domain-containing protein SUUR. Staining was retained in SUUR knock-out mutants but lost upon overexpression of SUUR. Somatic cells in Macrostomum lignano were strongly labeled, but pluripotent stem cells labeled weakly. Similarly, germline stem cells in Drosophila ovaries were weakly labeled compared to most other cells. The unexpected presence of G4 structures in heterochromatin and the difference in G4 staining between somatic cells and stem cells with germline DNA in ciliates, flatworms, flies and mammals point to a conserved role for G4 structures in nuclear organization and cellular differentiation.
Topics: Animals; Ciliophora; Drosophila; G-Quadruplexes; Germ Cells; Guanine; Heterochromatin; Histones; Islets of Langerhans; Platyhelminths; Polytene Chromosomes; Rats
PubMed: 26384414
DOI: 10.1093/nar/gkv900 -
Scientific Reports Apr 2021Micronucleoli are among the structures composing the peculiar scenario of the nucleolus in salivary gland nuclei of dipterans representative of Sciaridae. Micronucleolar...
Micronucleoli are among the structures composing the peculiar scenario of the nucleolus in salivary gland nuclei of dipterans representative of Sciaridae. Micronucleolar bodies contain ribosomal DNA and RNA, are transcriptionally active and may appear free in the nucleoplasm or associated with specific chromosome regions in salivary gland nuclei. This report deals with an extreme case of nucleolar fragmentation/dispersion detected in the salivary gland of Schwenkfeldina sp. Such a phenomenon in this species was found to be restricted to cell types undergoing polyteny and seems to be differentially controlled according to the cell type. Furthermore, transcriptional activity was detected in virtually all the micronucleolar bodies generated in the salivary gland. The relative proportion of the rDNA in polytene and diploid tissues showed that rDNA under-replication did not occur in polytene nuclei suggesting that the nucleolar and concomitant rDNA dispersion in Schwenkfeldina sp. may reflect a previously hypothesised process in order to counterbalance the rDNA loss due to the under-replication. The chromosomal distribution of epigenetic markers for the heterochromatin agreed with early cytological observations in this species suggesting that heterochromatin is spread throughout the chromosome length of Schwenkfeldina sp. A comparison made with results from another sciarid species argues for a role played by the heterochromatin in the establishment of the rDNA topology in polytene nuclei of Sciaridae.
Topics: Animals; Cell Nucleolus; DNA Fragmentation; DNA Replication; DNA, Ribosomal; Diptera; Heterochromatin; Polytene Chromosomes; RNA, Ribosomal; Salivary Glands; Transcription, Genetic
PubMed: 33863925
DOI: 10.1038/s41598-021-87012-5 -
Chromosome Research : An International... Dec 2022Studying the probability distribution of replication initiation along a chromosome is a huge challenge. Drosophila polytene chromosomes in combination with...
Studying the probability distribution of replication initiation along a chromosome is a huge challenge. Drosophila polytene chromosomes in combination with super-resolution microscopy provide a unique opportunity for analyzing the probabilistic nature of replication initiation at the ultrastructural level. Here, we developed a method for synchronizing S-phase induction among salivary gland cells. An analysis of the replication label distribution in the first minutes of S phase and in the following hours after the induction revealed the dynamics of replication initiation. Spatial super-resolution structured illumination microscopy allowed identifying multiple discrete replication signals and to investigate the behavior of replication signals in the first minutes of the S phase at the ultrastructural level. We identified replication initiation zones where initiation occurs stochastically. These zones differ significantly in the probability of replication initiation per time unit. There are zones in which initiation occurs on most strands of the polytene chromosome in a few minutes. In other zones, the initiation on all strands takes several hours. Compact bands are free of replication initiation events, and the replication runs from outer edges to the middle, where band shapes may alter.
Topics: Animals; Drosophila; Polytene Chromosomes; Microscopy; DNA Replication; Chromosomes; Drosophila melanogaster
PubMed: 35226231
DOI: 10.1007/s10577-021-09679-w -
Annual Review of Biochemistry 2015Eukaryotic gene expression is the result of the integrated action of multimolecular machineries. These machineries associate with gene transcripts, often already nascent... (Review)
Review
Eukaryotic gene expression is the result of the integrated action of multimolecular machineries. These machineries associate with gene transcripts, often already nascent precursor messenger RNAs (pre-mRNAs). They rebuild the transcript and convey properties allowing the processed transcript, the mRNA, to be exported to the cytoplasm, quality controlled, stored, translated, and degraded. To understand these integrated processes, one must understand the temporal and spatial aspects of the fate of the gene transcripts in relation to interacting molecular machineries. Improved methodology is necessary to study gene expression in vivo for endogenous genes. A complementary approach is to study biological systems that provide exceptional experimental possibilities. We describe such a system, the Balbiani ring (BR) genes in polytene cells in the dipteran Chironomus tentans. The BR genes, along with their pre-mRNA-protein complexes (pre-mRNPs) and mRNA-protein complexes (mRNPs), allow the visualization of intact cell nuclei and enable analyses of where and when different molecular machineries associate with and act on the BR pre-mRNAs and mRNAs.
Topics: Active Transport, Cell Nucleus; Animals; Cell Nucleus; Chironomidae; Chromosomal Puffs; Genes, Insect; Insect Proteins; RNA Processing, Post-Transcriptional; Ribonucleoproteins
PubMed: 26034888
DOI: 10.1146/annurev-biochem-060614-034150 -
Cells Mar 2023Nrf2 is the dominant cellular stress response factor that protects cells through transcriptional responses to xenobiotic and oxidative stimuli. Nrf2 malfunction is...
Nrf2 is the dominant cellular stress response factor that protects cells through transcriptional responses to xenobiotic and oxidative stimuli. Nrf2 malfunction is highly correlated with many human diseases, but the underlying molecular mechanisms remain to be fully uncovered. GATA4 is a conserved GATA family transcription factor that is essential for cardiac and dorsal epidermal development. Here, we describe a novel interaction between Nrf2 and GATA4 proteins, i.e., cap'n'collar C (CncC) and Pannier (Pnr), respectively. Using the bimolecular fluorescence complementation (BiFC) assay-a unique imaging tool for probing protein complexes in living cells-we detected CncC-Pnr complexes in the nuclei of embryonic and salivary gland cells. Visualization of CncC-Pnr BiFC signals on the polytene chromosome revealed that CncC and Pnr tend to form complexes in euchromatic regions, with a preference for loci that are not highly occupied by CncC or Pnr alone. Most genes within these loci are activated by the CncC-Pnr BiFC, but not by individually expressed CncC or Pnr fusion proteins, indicating a novel mechanism whereby CncC and Pnr interact at specific genomic loci and coactivate genes at these loci. Finally, CncC-induced early lethality can be rescued by Pnr depletion, suggesting that CncC and Pnr function in the same genetic pathway during the early development of . Taken together, these results elucidate a novel crosstalk between the Nrf2 xenobiotic/oxidative response factor and GATA factors in the transcriptional regulation of development. This study also demonstrates that the polytene chromosome BiFC assay is a valuable tool for mapping genes that are targeted by specific transcription factor complexes.
Topics: Animals; Chromatin; Drosophila; Drosophila Proteins; GATA4 Transcription Factor; NF-E2-Related Factor 2; Polytene Chromosomes; Xenobiotics; Transcriptional Activation
PubMed: 36980279
DOI: 10.3390/cells12060938 -
Cell Nov 2015Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed...
Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to 10-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes.
Topics: Animals; Cell Nucleus; Chromosomal Puffs; Diploidy; Drosophila melanogaster; Genetic Techniques; Larva; Polytene Chromosomes
PubMed: 26544940
DOI: 10.1016/j.cell.2015.10.026 -
Genetica Oct 2014Comparative genomics in Drosophila began in 1940, when Muller stated that the ancestral haploid karyotype of this genus is constituted by five acrocentric chromosomes...
Comparative genomics in Drosophila began in 1940, when Muller stated that the ancestral haploid karyotype of this genus is constituted by five acrocentric chromosomes and one dot chromosome, named A to F elements. In some species of the willistoni group such as Drosophila willistoni and D. insularis, the F element, instead of a dot chromosome, has been incorporated into the E element, forming chromosome III (E + F fusion). The aim of this study was to investigate the scope of the E + F fusion in the willistoni group, evaluating six other species. Fluorescent in situ hybridization was used to locate two genes of the F element previously studied-cubitus interruptus (ci) and eyeless (ey)-in species of the willistoni and bocainensis subgroups. Moreover, polytene chromosome photomaps corresponding to the F element (basal portion of chromosome III) were constructed for each species studied. In D. willistoni, D. paulistorum and D. equinoxialis, the ci gene was located in subSectction 78B and the ey gene in 78C. In D. tropicalis, ci was located in subSection 76B and ey in 76C. In species of the bocainensis subgroup, ci and ey were localized, respectively, at subsections 76B and 76C in D. nebulosa and D. capricorni, and 76A and 76C in D. fumipennis. Despite the differences in the subsection numbers, all species showed the same position for ci and ey. The results confirm the synteny of E + F fusion in willistoni and bocainensis subgroups, and allow estimating the occurrence of this event at 15 Mya, at least.
Topics: Animals; Chromosome Mapping; Chromosomes, Insect; DNA-Binding Proteins; Drosophila; Drosophila Proteins; Evolution, Molecular; Genes, Insect; In Situ Hybridization, Fluorescence; Microscopy, Fluorescence; Polytene Chromosomes; Species Specificity; Synteny; Time Factors; Transcription Factors
PubMed: 25134938
DOI: 10.1007/s10709-014-9784-3 -
Scientific Reports Nov 2018Establishment and maintenance of histone acetylation levels are critical for metazoan development and viability. Disruption of the balance between acetylation and...
Establishment and maintenance of histone acetylation levels are critical for metazoan development and viability. Disruption of the balance between acetylation and deacetylation by treatment with chemical histone deacetylase (HDAC) inhibitors results in loss of cell proliferation, differentiation and/or apoptosis. Histone deacetylation by the SIN3 complex is essential in Drosophila and mice, as loss of the scaffolding factor SIN3 or the associated HDAC results in lethality. The objective of this study is to elucidate contributions of SIN3 complex components to these essential processes. We used the Drosophila model organism to carry out a systematic functional analysis of the SIN3 complex. We find that SIN3 associated proteins are essential for viability and cell proliferation during development. Additionally, tissue specific reduction of SIN3 complex components results in abnormal wing development. Interestingly, while knockdown of each factor resulted in similar phenotypes, their individual effects on recruitment of SIN3 to polytene chromosomes are distinct. Reduction of some factors leads to large changes in the morphology of the chromosome and/or greatly reduced SIN3 binding. These findings suggest that while individual SIN3 complex components work through distinct molecular mechanisms, they each make a substantial contribution to the overall function of this highly conserved histone deacetylase complex.
Topics: Animals; Cell Proliferation; Cell Survival; Drosophila Proteins; Drosophila melanogaster; Female; Histones; Male; Sin3 Histone Deacetylase and Corepressor Complex
PubMed: 30451916
DOI: 10.1038/s41598-018-35093-0