Review

Authors: Kristen M Johansen, Weili Cai, Huai Deng...

The giant polytene chromosomes from Drosophila third instar larval salivary glands provide an important model system for studying the architectural changes in chromatin morphology associated with the process of transcription initiation and elongation. Especially, analysis of the heat shock response has proved useful in correlating chromatin structure remodeling with transcriptional activity. An important tool for such studies is the labeling of polytene chromosome squash preparations with antibodies to the enzymes, transcription factors, or histone modifications of interest. However, in any immunohistochemical experiment there will be advantages and disadvantages to different methods of fixation and sample preparation, the relative merits of which must be balanced. Here we provide detailed protocols for polytene chromosome squash preparation and discuss their relative pros and cons in terms of suitability for reliable antibody labeling and preservation of high resolution chromatin structure.

Topics: Animals; Chromatin; Chromosomes; Drosophila; Epigenesis, Genetic; Immunoenzyme Techniques; Transcription, Genetic

PubMed: 19272452
DOI: 10.1016/j.ymeth.2009.02.019

Authors: Nicholas Allen Kinney, Igor V Sharakhov, Alexey V Onufriev...

BACKGROUND

It is well recognized that the interphase chromatin of higher eukaryotes folds into non-random configurations forming territories within the nucleus. Chromosome territories have biologically significant properties, and understanding how these properties change with time during lifetime of the cell is important. Chromosome-nuclear envelope (Chr-NE) interactions play a role in epigenetic regulation of DNA replication, repair, and transcription. However, their role in maintaining chromosome territories remains unclear.

RESULTS

We use coarse-grained molecular dynamics simulations to study the effects of Chr-NE interactions on the dynamics of chromosomes within a model of the Drosophila melanogaster regular (non-polytene) interphase nucleus, on timescales comparable to the duration of interphase. The model simulates the dynamics of chromosomes bounded by the NE. Initially, the chromosomes in the model are prearranged in fractal-like configurations with physical parameters such as nucleus size and chromosome persistence length taken directly from experiment. Time evolution of several key observables that characterize the chromosomes is quantified during each simulation: chromosome territories, chromosome entanglement, compactness, and presence of the Rabl (polarized) chromosome arrangement. We find that Chr-NE interactions help maintain chromosome territories by slowing down and limiting, but not eliminating, chromosome entanglement on biologically relevant timescales. At the same time, Chr-NE interactions have little effect on the Rabl chromosome arrangement as well as on how chromosome compactness changes with time. These results are rationalized by simple dimensionality arguments, robust to model details. All results are robust to the simulated activity of topoisomerase, which may be present in the interphase cell nucleus.

CONCLUSIONS

Our study demonstrates that Chr-NE attachments may help maintain chromosome territories, while slowing down and limiting chromosome entanglement on biologically relevant timescales. However, Chr-NE attachments have little effect on chromosome compactness or the Rabl chromosome arrangement.

Topics: Animals; Chromosomes, Insect; Drosophila melanogaster; Interphase; Models, Molecular; Nuclear Envelope; Polytene Chromosomes

PubMed: 29357905
DOI: 10.1186/s13072-018-0173-5

Review

Authors: Jonathan Hodgkin

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

Review

Authors: Benjamin M Stormo, Donald T Fox

In this era of high-resolution mapping of chromosome territories, topological interactions, and chromatin states, it is increasingly appreciated that the positioning of chromosomes and their interactions within the nucleus is critical for cellular function. Due to their large size and distinctive structure, polytene chromosomes have contributed a wealth of knowledge regarding chromosome regulation. In this review, we discuss the diversity of polytene chromosomes in nature and in disease, examine the recurring structural features of polytene chromosomes in terms of what they reveal about chromosome biology, and discuss recent advances regarding how polytene chromosomes are assembled and disassembled. After over 130 years of study, these giant chromosomes are still powerful tools to understand chromosome biology.

Topics: Animals; DNA Replication; Disease Susceptibility; Gene Expression Regulation; Gene-Environment Interaction; Genetic Loci; Genetics; Polyploidy; Polytene Chromosomes; Research

PubMed: 28779272
DOI: 10.1007/s10577-017-9562-z

Authors: D F Eberl, L A Perkins, M Engelstein...

Polytene section 17 of the X chromosome of Drosophila melanogaster, previously known to contain six putative lethal complementation groups important in oogenesis and embryogenesis, has here been further characterized genetically and developmentally. We constructed fcl+Y, a duplication of this region, which allowed us to conduct mutagenesis screens specific for the region and to perform complementation analyses (previously not possible). We recovered 67 new lethal mutations which defined 15 complementation groups within Df(1)N19 which deletes most of polytene section 17. The zygotic lethal phenotypes of these and preexisting mutations within polytene section 17 were examined, and their maternal requirements were analysed in homozygous germline clones using the dominant female sterile technique. We present evidence that an additional gene, which produces two developmentally regulated transcripts, is located in this region and is involved in embryogenesis, although no mutations in this gene were identified. In this interval of 37 to 43 polytene chromosome bands we have defined 17 genes, 12 (71%) of which are of significance to oogenesis or embryogenesis.

Topics: Amino Acid Sequence; Animals; Chromosome Banding; Cloning, Molecular; Crosses, Genetic; Drosophila melanogaster; Female; Genetic Complementation Test; Male; Molecular Sequence Data; Mutagenesis; Phenotype; Restriction Mapping; Salivary Glands; Transformation, Genetic; X Chromosome

PubMed: 1551578
DOI: 10.1093/genetics/130.3.569

Authors: Darya S Sidorenko, Ivan A Sidorenko, Tatyana Yu Zykova...

The fourth chromosome smallest in the genome of Drosophila melanogaster differs from other chromosomes in many ways. It has high repeat density in conditions of a large number of active genes. Gray bands represent a significant part of this polytene chromosome. Specific proteins including HP1a, POF, and dSETDB1 establish the epigenetic state of this unique chromatin domain. In order to compare maps of localization of genes, bands, and chromatin types of the fourth chromosome, we performed FISH analysis of 38 probes chosen according to the model of four chromatin types. It allowed clarifying the dot chromosome cytological map consisting of 16 loose gray bands, 11 dense black bands, and 26 interbands. We described the relation between chromatin states and bands. Open aquamarine chromatin mostly corresponds to interbands and it contains 5'UTRs of housekeeping genes. Their coding parts are embedded in gray bands substantially composed of lazurite chromatin of intermediate compaction. Polygenic black bands contain most of dense ruby chromatin, and also some malachite and lazurite. Having an accurate map of the fourth chromosome bands and its correspondence to physical map, we found that DNase I hypersensitivity sites, ORC2 protein, and P-elements are mainly located in open aquamarine chromatin, while element 1360, characteristic of the fourth chromosome, occupies band chromatin types. POF and HP1a proteins providing special organization of this chromosome are mostly located in aquamarine and lazurite chromatin. In general, band organization of the fourth chromosome shares the features of the whole Drosophila genome.

Topics: Animals; Chromosome Banding; Chromosomes, Insect; Drosophila Proteins; Drosophila melanogaster; Female; Genome, Insect; Male; Polytene Chromosomes

PubMed: 31041520
DOI: 10.1007/s00412-019-00703-x

Authors: John Sedat, Angus McDonald, Hu Cang...

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

Authors: Phillip George, Atashi Sharma, Igor V Sharakhov...

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

Authors: Eduardo Gorab

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

Authors: Ann Jacob Stocker, Michele Schiffer, Eduardo Gorab...

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