Fly Chromosomes Research Articles

Low dose rate γ-irradiation protects fruit fly chromosomes from double strand breaks and telomere fusions by reducing the esi-RNA biogenesis factor Loquacious

It is still continuously debated whether the low-dose/dose-rate (LDR) of ionizing radiation represents a hazard for humans. Model organisms, such as fruit flies, are considered valuable systems to reveal insights into this issue. We found that, in wild-type Drosophila melanogaster larval neuroblasts, the frequency of Chromosome Breaks (CBs), induced by acute γ-irradiation, is considerably reduced when flies are previously exposed to a protracted dose of 0.4 Gy delivered at a dose rate of 2.5 mGy/h. This indicates that this exposure, which is associated with an increased expression of DNA damage response proteins, induces a radioadaptive response (RAR) that protects Drosophila from extensive DNA damage. Interestingly, the same exposure reduces the frequency of telomere fusions (TFs) from Drosophila telomere capping mutants suggesting that the LDR can generally promote a protective response on chromatin sites that are recognized as DNA breaks. Deep RNA sequencing revealed that RAR is associated with a reduced expression of Loquacious D (Loqs-RD) gene that encodes a well-conserved dsRNA binding protein required for esiRNAs biogenesis. Remarkably, loss of Loqs mimics the LDR-mediated chromosome protection as it decreases the IR-induced CBs and TFs frequency. Thus, our molecular characterization of RAR identifies Loqs as a key factor in the cellular response to LDR and in the epigenetic routes involved in radioresistance.

The analysis of mutant alleles of different strength reveals multiple functions of topoisomerase 2 in regulation of Drosophila chromosome structure.

Topoisomerase II is a major component of mitotic chromosomes but its role in the assembly and structural maintenance of chromosomes is rather controversial, as different chromosomal phenotypes have been observed in various organisms and in different studies on the same organism. In contrast to vertebrates that harbor two partially redundant Topo II isoforms, Drosophila and yeasts have a single Topo II enzyme. In addition, fly chromosomes, unlike those of yeast, are morphologically comparable to vertebrate chromosomes. Thus, Drosophila is a highly suitable system to address the role of Topo II in the assembly and structural maintenance of chromosomes. Here we show that modulation of Top2 function in living flies by means of mutant alleles of different strength and in vivo RNAi results in multiple cytological phenotypes. In weak Top2 mutants, meiotic chromosomes of males exhibit strong morphological abnormalities and dramatic segregation defects, while mitotic chromosomes of larval brain cells are not affected. In mutants of moderate strength, mitotic chromosome organization is normal, but anaphases display frequent chromatin bridges that result in chromosome breaks and rearrangements involving specific regions of the Y chromosome and 3L heterochromatin. Severe Top2 depletion resulted in many aneuploid and polyploid mitotic metaphases with poorly condensed heterochromatin and broken chromosomes. Finally, in the almost complete absence of Top2, mitosis in larval brains was virtually suppressed and in the rare mitotic figures observed chromosome morphology was disrupted. These results indicate that different residual levels of Top2 in mutant cells can result in different chromosomal phenotypes, and that the effect of a strong Top2 depletion can mask the effects of milder Top2 reductions. Thus, our results suggest that the previously observed discrepancies in the chromosomal phenotypes elicited by Topo II downregulation in vertebrates might depend on slight differences in Topo II concentration and/or activity.

Imaging Through the Pupal Case of <em>Drosophila melanogaster</em>

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 µm of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of <em>Drosophila melanogaster</em>

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 µm of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Population Structure and the Colonization Route of One of the Oldest North American Invasive Insects: Stories from the Worn Road of the Hessian Fly, Mayetiola destructor (Say)

An integral part to understanding the biology of an invasive species is determining its origin, particularly in pest species. As one of the oldest known invasive species, the goals of this study were to evaluate the evidence of a westward expansion of Hessian fly into North America, from a potential singular introduction event, and the population genetic structure of current populations. Levels of genetic diversity and population structure in the Hessian fly were compared across North America, Europe, North Africa, Western Asia, and New Zealand. Furthermore, Old World populations were evaluated as possible sources of introduction. We tested diversity and population structure by examining 18 microsatellite loci with coverage across all four Hessian fly chromosomes. Neither genetic diversity nor population genetic structure provided evidence of a westward movement from a single introduction in North America. Introduced populations in North America did not show identity or assignment to any Old World population, likely indicating a multiple introduction scenario with subsequent gene flow between populations. Diversity and selection were assessed on a chromosomal level, with no differences in diversity or selection between chromosomes or between native and introduced populations.

Regions of Unusually High Flexibility Occur Frequently in Human Genomic DNA

Remarkable progress has been made in genome science during the past decade, but understanding of genomes of eukaryotes is far from complete. We have created DNA flexibility maps of the human, mouse, fruit fly, and nematode chromosomes. The maps revealed that all of these chromosomes have markedly flexible DNA regions (We named them SPIKEs). SPIKEs occur more frequently in the human chromosomes than in the mouse, fruit fly, and nematode chromosomes. Markedly rigid DNA regions (rSPIKEs) are also present in these chromosomes. The ratio of the number of SPIKEs to the total number of SPIKEs and rSPIKEs correlated positively with evolutionary stage among the organisms. Repetitive DNA sequences with flexible and rigid properties contribute to the formation of SPIKEs and rSPIKEs respectively. However, non-repetitive flexible and rigid sequences appear to play a major role in SPIKE and rSPIKE formation respectively. They might be involved in the genome-folding mechanism of eukaryotes.

Chromatin remodeling in the aging genome of Drosophila

Chromatin structure affects the accessibility of DNA to transcription, repair, and replication. Changes in chromatin structure occur during development, but less is known about changes during aging. We examined the state of chromatin structure and its effect on gene expression during aging in Drosophila at the whole genome and cellular level using whole-genome tiling microarrays of activation and repressive chromatin marks, whole-genome transcriptional microarrays and single-cell immunohistochemistry. We found dramatic reorganization of chromosomal regions with age. Mapping of H3K9me3 and HP1 signals to fly chromosomes reveals in young flies the expected high enrichment in the pericentric regions, the 4th chromosome, and islands of facultative heterochromatin dispersed throughout the genome. With age, there is a striking reduction in this enrichment resulting in a nearly equivalent level of H3K9me3 and HP1 in the pericentric regions, the 4th chromosome, facultative heterochromatin, and euchromatin. These extensive changes in repressive chromatin marks are associated with alterations in age-related gene expression. Large-scale changes in repressive marks with age are further substantiated by single-cell immunohistochemistry that shows changes in nuclear distribution of H3K9me3 and HP1 marks with age. Such epigenetic changes are expected to directly or indirectly impinge upon important cellular functions such as gene expression, DNA repair, and DNA replication. The combination of genome-wide approaches such as whole-genome chromatin immunoprecipitation and transcriptional studies in conjunction with single-cell immunohistochemistry as shown here provide a first step toward defining how changes in chromatin may contribute to the process of aging in metazoans.

A physically anchored genetic map and linkage to avirulence reveals recombination suppression over the proximal region of Hessian fly chromosome A2.

Resistance in wheat (Triticum aestivum) to the Hessian fly (Mayetiola destructor), a major insect pest of wheat, is based on a gene-for-gene interaction. Close linkage (3 +/- 2 cM) was discovered between Hessian fly avirulence genes vH3 and vH5. Bulked segregant analysis revealed two DNA markers (28-178 and 23-201) within 10 cM of these loci and only 3 +/- 2 cM apart. However, 28-178 was located in the middle of the short arm of Hessian fly chromosome A2 whereas 23-201 was located in the middle of the long arm of chromosome A2, suggesting the presence of severe recombination suppression over its proximal region. To further test that possibility, an AFLP-based genetic map of the Hessian fly genome was constructed. Fluorescence in situ hybridization of 20 markers on the genetic map to the polytene chromosomes of the Hessian fly indicated good correspondence between the linkage groups and the four Hessian fly chromosomes. The physically anchored genetic map is the first of any gall midge species. The proximal region of mitotic chromosome A2 makes up 30% of its length but corresponded to <3% of the chromosome A2 genetic map.

A Gain-of-Function Screen for Genes That Affect the Development of the Drosophila Adult External Sensory Organ.

IN natural populations of Drosophila melanogaster throughout the world, a minority of individuals are usually infected by the Rhabdovirus sigma. This virus does not integrate into the fly chromosomes and is only vertically transmitted through male and female gametes ([Fleuriet 1999][1]). Of the fly

Preparation of House Fly Chromosomes

Over the years the adult house fly, Musca domestica L., has shown itself to be an excellent experimental animal in various fields of entomological research. Since some research is of a cytological nature, a need exists for additional techniques for studying this insect. Recently, French et al. (1962) published a method for preparing chromosomes from the brains of mosquito larvae and from the ovaries and testes of mosquito pupae. Oster and Balaban (1963) published a method of preparing somatic chromosomes from the cerebral ganglion of Drosophila larvae and also demonstrated that this method would work on the cerebral ganglion of house fly larvae. By modifying these procedures we developed a technique that enabled us to obtain consistently good chromosome preparations from the gonads of adult male house flies.