-
Journal of Visualized Experiments : JoVE Sep 2012Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important...
Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions. Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti and Cx. quinquefasciatus, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti, the accumulation of multiple chromosomal rearrangements in cell line chromosomes makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.
Topics: Animals; Chromosomes, Insect; Culicidae; Female; In Situ Hybridization, Fluorescence; Mitosis
PubMed: 23007640
DOI: 10.3791/4215 -
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 -
Memorias Do Instituto Oswaldo Cruz Jul 2002Drosophila mediopunctata belongs to the tripunctata group, and is one of the commonest Drosophila species collected in some places in Brazil, especially in the winter. A...
Drosophila mediopunctata belongs to the tripunctata group, and is one of the commonest Drosophila species collected in some places in Brazil, especially in the winter. A standard map of the polytene chromosomes is presented. The breakpoints of the naturally occurring chromosomal rearrangements are marked on the map. The distribution of breaking points through the chromosomes of D. mediopunctata is apparently non-random. Chromosomes X, II and IV show inversion polymorphisms. Chromosome II is the most polymorphic, with 17 inversions, 8 inversions in the distal region and 9 in the proximal region. Chromosome X has four different gene arrangements, while chromosome IV has only two.
Topics: Animals; Chromosome Inversion; Chromosome Mapping; Drosophila; Drosophila Proteins; Female; Polymorphism, Genetic
PubMed: 12219137
DOI: 10.1590/s0074-02762002000500019 -
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 -
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 -
Genetics Sep 2013Dynamic regulation of chromosome structure and organization is critical for fundamental cellular processes such as gene expression and chromosome segregation. Condensins...
Dynamic regulation of chromosome structure and organization is critical for fundamental cellular processes such as gene expression and chromosome segregation. Condensins are conserved chromosome-associated proteins that regulate a variety of chromosome dynamics, including axial shortening, lateral compaction, and homolog pairing. However, how the in vivo activities of condensins are regulated and how functional interactors target condensins to chromatin are not well understood. To better understand how Drosophila melanogaster condensin is regulated, we performed a yeast two-hybrid screen and identified the chromo-barrel domain protein Mrg15 to interact with the Cap-H2 condensin subunit. Genetic interactions demonstrate that Mrg15 function is required for Cap-H2-mediated unpairing of polytene chromosomes in ovarian nurse cells and salivary gland cells. In diploid tissues, transvection assays demonstrate that Mrg15 inhibits transvection at Ubx and cooperates with Cap-H2 to antagonize transvection at yellow. In cultured cells, we show that levels of chromatin-bound Cap-H2 protein are partially dependent on Mrg15 and that Cap-H2-mediated homolog unpairing is suppressed by RNA interference depletion of Mrg15. Thus, maintenance of interphase chromosome compaction and homolog pairing status requires both Mrg15 and Cap-H2. We propose a model where the Mrg15 and Cap-H2 protein-protein interaction may serve to recruit Cap-H2 to chromatin and facilitates compaction of interphase chromatin.
Topics: Adenosine Triphosphatases; Animals; Chromatin; Chromosomal Proteins, Non-Histone; Chromosome Pairing; DNA-Binding Proteins; Drosophila; Drosophila Proteins; Epigenesis, Genetic; Homeodomain Proteins; Interphase; Multiprotein Complexes; Polytene Chromosomes; Protein Binding; Transcription Factors
PubMed: 23821596
DOI: 10.1534/genetics.113.153544 -
ELife Dec 2022Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early...
Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underreplicated. SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here, we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, or ) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier, and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins.
Topics: Animals; Drosophila; DNA-Binding Proteins; Heterochromatin; Drosophila melanogaster; Drosophila Proteins; Euchromatin; Chromatin; DNA Replication
PubMed: 36458689
DOI: 10.7554/eLife.81828 -
G3 (Bethesda, Md.) Aug 2017Eukaryote genomes are replete with repetitive DNAs. This class includes tandemly repeated satellite DNAs (satDNA) which are among the most abundant, fast evolving (yet...
Eukaryote genomes are replete with repetitive DNAs. This class includes tandemly repeated satellite DNAs (satDNA) which are among the most abundant, fast evolving (yet poorly studied) genomic components. Here, we used high-throughput sequencing data from three cactophilic species, , , and , to access and study their whole satDNA landscape. In total, the software identified five satDNAs, three previously described (, and ) and two novel ones ( and ). Only is shared among all three species. The satDNA repeat length falls within only two classes, between 130 and 200 bp or between 340 and 390 bp. FISH on metaphase and polytene chromosomes revealed the presence of satDNA arrays in at least one of the following genomic compartments: centromeric, telomeric, subtelomeric, or dispersed along euchromatin. The chromosomal distribution ranges from a single chromosome to almost all chromosomes of the complement. Fiber-FISH and sequence analysis of contigs revealed interspersion between and in the microchromosomes of Phylogenetic analyses showed that the pBuM satDNA underwent concerted evolution at both interspecific and intraspecific levels. Based on RNA-seq data, we found transcription activity for (in ) and (in ) in all five analyzed developmental stages, most notably in pupae and adult males. Our data revealed that cactophilic present the lowest amount of satDNAs (1.9-2.9%) within the genus reported so far. We discuss how our findings on the satDNA location, abundance, organization, and transcription activity may be related to functional aspects.
Topics: Animals; Cactaceae; Centromere; DNA Probes; DNA Transposable Elements; DNA, Satellite; Drosophila; Evolution, Molecular; Genome, Insect; In Situ Hybridization, Fluorescence; Phylogeny; Polytene Chromosomes; Repetitive Sequences, Nucleic Acid; Sequence Analysis, DNA; Species Specificity; Telomere; Transcription, Genetic
PubMed: 28659292
DOI: 10.1534/g3.117.042093 -
Nucleic Acids Research Oct 1997GAGA factor (GAF) binds to specific DNA sequences and participates in a complex spectrum of chromosomal activities. Products of the Trithorax-like locus (Trl), which... (Review)
Review
GAGA factor (GAF) binds to specific DNA sequences and participates in a complex spectrum of chromosomal activities. Products of the Trithorax-like locus (Trl), which encodes multiple GAF isoforms, are required for homeotic gene expression and are essential for Drosophila development. While homozygous null mutations in Trl are lethal, heterozygotes display enhanced position effect variegation (PEV) indicative of the broad role of GAF in chromatin architecture and its positive role in gene expression.The distribution of GAF on chromosomes is complex, as it is associated with hundreds of chromosomal loci in euchromatin of salivary gland polytene chromosomes, however, it also displays a strong association with pericentric heterochromatin in diploid cells, where it appears to have roles in chromosome condensation and segregation. At higher resolution GAF binding sites have been identified in the regulatory regions of many genes. In some cases, the positive role of GAF in gene expression has been examined in detail using a variety of genetic, biochemical, and cytological approaches. Here we review what is currently known of GAF and, in the context of the heat shock genes of Drosophila, we examine the effects of GAF on multiple steps in gene expression.
Topics: Animals; Chromatin; DNA; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Drosophila Proteins; Gene Expression Regulation; Heat Shock Transcription Factors; Heat-Shock Proteins; Homeodomain Proteins; Transcription Factors
PubMed: 9321643
DOI: 10.1093/nar/25.20.3963