Drosophila Chromosomes Research Articles

RNA-Guided Nucleases: A New Era for Engineering the Genomes of Model and Nonmodel Organisms

In 1946, H. J. Muller was awarded the Nobel Prize in Medicine for his discovery with Drosophila melanogaster , some 20 years earlier, that exposure to X-rays caused mutation ([Muller 1927][1]). This led to the identification of a large number of Drosophila mutants and chromosome rearrangements, many

Structural features of 3C6/C7 interband chromatin organization in Drosophila melanogaster polytene chromosomes

Mechanisms of chromatin decompaction in interbands of Drosophila polytene chromosomes have been studied. Using the example of interband 3C6/C7 of the X chromosome, we investigated the ability of different DNA segments to form an interband in a new genetic environment. We applied site-specific FLP recombination between two transposons with FRT-sites that allows introducing the DNA fragments from the interband 3C6/C7 into pICon(dv) transposon located in cytologically well-characterized 84F region of chromosome 3 followed by electron microscopic analysis of changes in the region caused by insertion of the DNA fragments into the transposon. It was shown that the insertion of a 276-bp DNA fragment from the 3C6/C7 region into the pICon(dv) transposon leads to the formation of a new interband between two thin bands represented by the transposon material. This DNA fragment is the known minimal sequence that is necessary and sufficient for interband generation. In addition, the sequence containing three copies repeated in tandem of 0.9 kb DNA from the interband 3C6/C7, including the 276-bp fragment, were integrated in the transposon. The presence of introduced DNA fragments did not change the morphology of the resulting interband. It was shown that the sites of DNase I hypersensitivity were saved in transposon sequences introduced into a new genetic environment. The data obtained allow analysis to be started of specific factors (proteins, DNA motifs, etc.) that determine the formation of decompacted chromatin in a certain interband region and chromomeric organization of interphase chromosomes in Drosophila as a whole.

Reversal of an ancient sex chromosome to an autosome in Drosophila.

Although transitions of sex-determination mechanisms are frequent in species with homomorphic sex chromosomes, heteromorphic sex chromosomes are thought to represent a terminal evolutionary stage owing to chromosome-specific adaptations such as dosage compensation or an accumulation of sex-specific mutations. Here we show that an autosome of Drosophila, the dot chromosome, was ancestrally a differentiated X chromosome. We analyse the whole genome of true fruitflies (Tephritidae), flesh flies (Sarcophagidae) and soldier flies (Stratiomyidae) to show that genes located on the dot chromosome of Drosophila are X-linked in outgroup species, whereas Drosophila X-linked genes are autosomal. We date this chromosomal transition to early drosophilid evolution by sequencing the genome of other Drosophilidae. Our results reveal several puzzling aspects of Drosophila dot chromosome biology to be possible remnants of its former life as a sex chromosome, such as its minor feminizing role in sex determination or its targeting by a chromosome-specific regulatory mechanism. We also show that patterns of biased gene expression of the dot chromosome during early embryogenesis, oogenesis and spermatogenesis resemble that of the current X chromosome. Thus, although sex chromosomes are not necessarily evolutionary end points and can revert back to an autosomal inheritance, the highly specialized genome architecture of this former X chromosome suggests that severe fitness costs must be overcome for such a turnover to occur.

The effect of JIL-1 on position-effect variegation is proportional to the total amount of heterochromatin in the genome

In this study we have taken advantage of recent whole genome sequencing studies that have determined the DNA content in the heterochromatic regions of each Drosophila chromosome to directly correlate the effect on position-effect variegation of a pericentric insertion reporter line, 118E-10 with the total amount of heterochromatic DNA. Heterochromatic DNA levels were manipulated by adding or subtracting a Y chromosome as well as by the difference in the amount of pericentric heterochromatin between the X and Y chromosome. The results showed a direct, linear relationship between the amount of heterochromatic DNA in the genome and the expression of the w marker gene in the 118E-10 pericentric reporter line and that increasing amounts of heterochromatic DNA resulted in increasing amounts of pigment/gene activity. In Drosophila heterochromatic spreading and gene silencing is counteracted by H3S10 phosphorylation by the JIL-1 kinase, and we further demonstrate that the haplo-enhancer effect of JIL-1 is proportional to the amount of total heterochomatin, suggesting that JIL-1's activity is dynamically modulated to achieve a more or less constant balance depending on the levels of heterochromatic factors present.

Disengaging the Smc3/kleisin interface releases cohesin from Drosophila chromosomes during interphase and mitosis

Cohesin's Smc1, Smc3, and kleisin subunits create a tripartite ring within which sister DNAs are entrapped. Evidence suggests that DNA enters through a gate created by transient dissociation of the Smc1/3 interface. Release at the onset of anaphase is triggered by proteolytic cleavage of kleisin. Less well understood is the mechanism of release at other stages of the cell cycle, in particular during prophase when most cohesin dissociates from chromosome arms in a process dependent on the regulatory subunit Wapl. We show here that Wapl-dependent release from salivary gland polytene chromosomes during interphase and from neuroblast chromosome arms during prophase is blocked by translational fusion of Smc3's C-terminus to kleisin's N-terminus. Our findings imply that proteolysis-independent release of cohesin from chromatin is mediated by Wapl-dependent escape of DNAs through a gate created by transient dissociation of the Smc3/kleisin interface. Thus, cohesin's DNA entry and exit gates are distinct.

56 Large-scale alternation of nucleosome positioning potential in pericentromeric chromosome regions

It is difficult to obtain quantitative information on the protein bindings in the biological systems in the large-scale variant. We used nucleosome positioning potential (NPP) calculations on the basis of method published earlier (Fedoseyeva & Alexandrov, 2007). This method often demonstrates cluster character of NPP. Calculation of Furrier coefficients allows to analyze regional preferences of nucleosome binding. Often, the alternation of stronger and weaker binding regions takes place. Special interest deals with large-scale alteration when this may be interpreted in the terms of cellular biology. We determined them in pericentromeric regions of 2 and 3 chromosomes of Drosophila melanogaster. In one of the cases, sufficiently strong alternation of stronger and weaker NPP region was observed. The distance between nearby stronger ones is approximately 70 kbp. Fortunately, this observation may be interpreted by the competition of nucleosome with cohesin-type proteins for binding with sister chromatids. This binding maintains them side by side during appropriate stages in mitosis. The differences and likeness in NPP between left and right arms of chromosomes are discussed.

The Effect of Diazinon on Puffing Activity in Polytene Chromosomes of Drosophila melanogaster

This study investigates the effects of diazinon, an organic phosphoric insecticide, puffing on salivary gland polytene chromosomes of Drosophila melanogaster. Adult flies are applied diazinon and F1 generations were developed. The third instar larvae’s salivary glands polytene chromosomes were examined in F1 generation. Puff areas in polytene chromosomes are regions of RNA synthesis and provide information on interphase nucleus. Certain differences were detected in puffing model of polytene chromosomes following the diazinon application. These differences were either the retreat of certain active regions or the activation of

A Germline Clone Screen on theXChromosome Reveals Novel Meiotic Mutants inDrosophila melanogaster

In an effort to isolate novel meiotic mutants that are severely defective in chromosome segregation and/or exchange, we employed a germline clone screen of the X chromosome of Drosophila melanogaster. We screened over 120,000 EMS-mutagenized chromosomes and isolated 19 mutants, which comprised nine complementation groups. Four of these complementation groups mapped to known meiotic genes, including mei-217, mei-218, mei-9, and nod. Importantly, we have identified two novel complementation groups with strong meiotic phenotypes, as assayed by X chromosome nondisjunction. One complementation group is defined by three alleles, and the second novel complementation group is defined by a single allele. All 19 mutants are homozygous viable, fertile, and fully recessive. Of the 9 mutants that have been molecularly characterized, 5 are canonical EMS-induced transitions, and the remaining 4 are transversions. In sum, we have identified two new genes that are defined by novel meiotic mutants, in addition to isolating new alleles of mei-217, mei-218, mei-9, and nod.

Msl1-Mediated Dimerization of the Dosage Compensation Complex Is Essential for Male X-Chromosome Regulation in Drosophila

The Male-Specific Lethal (MSL) complex regulates dosage compensation of the male X chromosome in Drosophila. Here, we report the crystal structure of its MSL1/MSL2 core, where two MSL2 subunits bind to a dimer formed by two molecules of MSL1. Analysis of structure-based mutants revealed that MSL2 can only interact with the MSL1 dimer, but MSL1 dimerization is MSL2 independent. We show that Msl1 is a substrate for Msl2 E3 ubiquitin ligase activity. ChIP experiments revealed that Msl1 dimerization is essential for targeting and spreading of the MSL complex on X-linked genes; however, Msl1 binding to promoters of male and female cells is independent of the dimer status and other MSL proteins. Finally, we show that loss of Msl1 dimerization leads to male-specific lethality. We propose that Msl1-mediated dimerization of the entire MSL complex is required for Msl2 binding, X chromosome recognition, and spreading along the X chromosome.

Gene Density, Transcription, and Insulators Contribute to the Partition of the Drosophila Genome into Physical Domains

The mechanisms responsible for the establishment of physical domains in metazoan chromosomes are poorly understood. Here we find that physical domains in Drosophila chromosomes are demarcated at regions of active transcription and high gene density that are enriched for transcription factors and specific combinations of insulator proteins. Physical domains contain different types of chromatin defined by the presence of specific proteins and epigenetic marks, with active chromatin preferentially located at the borders and silenced chromatin in the interior. Domain boundaries participate in long-range interactions that may contribute to the clustering of regions of active or silenced chromatin in the nucleus. Analysis of transgenes suggests that chromatin is more accessible and permissive to transcription at the borders than inside domains, independent of the presence of active or silencing histone modifications. These results suggest that the higher-order physical organization of chromatin may impose an additional level of regulation over classical epigenetic marks.

Identification of Genes Underlying Hypoxia Tolerance inDrosophilaby a P-element Screen

Hypoxia occurs in physiologic conditions (e.g. high altitude) or during pathologic states (e.g. ischemia). Our research is focused on understanding the molecular mechanisms that lead to adaptation and survival or injury to hypoxic stress using Drosophila as a model system. To identify genes involved in hypoxia tolerance, we screened the P-SUP P-element insertion lines available for all the chromosomes of Drosophila. We screened for the eclosion rates of embryos developing under 5% O2 condition and the number of adult flies surviving one week after eclosion in the same hypoxic environment. Out of 2187 lines (covering ∼1870 genes) screened, 44 P-element lines representing 44 individual genes had significantly higher eclosion rates (i.e. >70%) than those of the controls (i.e. ∼7–8%) under hypoxia. The molecular function of these candidate genes ranged from cell cycle regulation, DNA or protein binding, GTP binding activity, and transcriptional regulators. In addition, based on pathway analysis, we found these genes are involved in multiple pathways, such as Notch, Wnt, Jnk, and Hedgehog. Particularly, we found that 20 out of the 44 candidate genes are linked to Notch signaling pathway, strongly suggesting that this pathway is essential for hypoxia tolerance in flies. By employing the UAS/RNAi-Gal4 system, we discovered that genes such as osa (linked to Wnt and Notch pathways) and lqf (Notch regulator) play an important role in survival and development under hypoxia in Drosophila. Based on these results and our previous studies, we conclude that hypoxia tolerance is a polygenic trait including the Notch pathway.

Induced transcription results in local changes in chromatin structure, replication timing, and DNA polytenization in a site of intercalary heterochromatin

In salivary gland polytene chromosomes of Drosophila melanogaster, the regions of intercalary heterochromatin are characterized by late replication, under-replication, and genetic silencing. Using Gal4/UAS system, we induced transcription of sequences adjacent to transgene insertions in the band 11A6-9. This activation resulted in a loss of "silent" and appearance of "active" epigenetic marks, recruitment of RNA polymerase II, and formation of a puff. The activated region is now early replicating and shows increased level of DNA polytenization. Notably, all these changes are restricted to the area around the inserts, whereas the rest of the band remains inactive and late replicating. Although only a short area near the insertion site is transcribed, it results in an "open" chromatin conformation in a much broader region. We conclude that regions of intercalary heterochromatin do not form stand-alone units of late replication and under-replication. Every part of such regions can be activated and polytenized independently of other parts.

An Assay to DetectIn VivoY Chromosome Loss inDrosophilaWing Disc Cells

Loss of the Y chromosome in Drosophila has no impact on cell viability and therefore allows us to assay the impact of environmental agents and genetic alterations on chromosomal loss. To detect in vivo chromosome loss in cells of the developing Drosophila wing primordia, we first engineered a Y chromosome with an attP docking site. By making use of the ΦC31 integrase system, we site-specifically integrated a genomic transgene encompassing the multiple wing hair (mwh) locus into this attP site, leading to a mwh+Y chromosome. This chromosome fully rescues the mwh mutant phenotype, an excellent recessive wing cell marker mutation. Loss of this mwh+Y chromosome in wing primordial cells then leads to manifestation of the mwh mutant phenotype in mwh-homozygous cells. The forming mwh clones permit us to quantify the effect of agents and genetic alterations by assaying frequency and size of the mwh mosaic spots. To illustrate the use of the mwh+Y loss system, the effects of four known mutagens (X-rays, colchicine, ethyl methanesulfonate, and formaldehyde) and two genetic conditions (loss- and gain-of-function lodestar mutant alleles) are documented. The procedure is simple, sensitive, and inexpensive.

Nuclear envelope attachment sites of interphase chromosomes: Barrier elements but not insulators

A neDNA fragment that protects a transgene from position effect variegation when flanking it was tested for insulator properties. The fragment did not act as an insulator. A similarity of neDNA and various barrier elements was examined, and Drosophila melanogaster and Mus musculus chromosome DNA regions homologous to neDNA were analyzed. Additional conserved sites and DNA duplex destabilization sites were found in the neDNA sequence, and DNA conformational specifics were assumed for the chromosomal neighborhood of neDNA sites of the D. melanogaster and M. musculus genomes.

The X chromosome, heterochromatin and speciation in malaria mosquitoes

The available data suggest that the X chromosome of Anopheles had originated independently from the X chromosome of Drosophila. In order to gain insights into a possible role of the X chromosome in speciation of malaria mosquitoes, we studied the pattern of rearrangements, genomic landscapes and heterochromatin variations among closely and distantly related species. • Our cytogenetic analysis of the An. gambiae species complex showed fixed differences in the amount and pattern of the X heterochromatin among sibling species as well as between the M and S forms (Fig. 1). Interestingly, the low-recombination pericentric region displayed the high level of polymorphism in the size of heterochromatin and the rDNA locus (Fig. 2). • The largest region of the high genomic divergence between the incipient species (the M and S forms) co-localizes with the heterochromatin of the An. gambiae X chromosome (Fig. 3). • Our comparative physical mapping of distantly related malaria mosquito species identified the fastest rate of inversion fixation on the X chromosome despite the paucity of inversion polymorphism (Fig. 4). • The analysis of the An. gambiae genome demonstrated the highest density of transposable elements, microsatellites, minisatellites, and satellites in the X chromosome (Fig. 5). • The X chromosome is enriched in genes that may be involved in premating isolation, such as genes encoding for proteins with molecular and signal transduction activity (Fig. 6). Thus, our study points out to the elevated evolutionary plasticity of the euchromatic gene order and heterochromatin structure of the X chromosome suggesting a possible role of the sex chromosome in speciation of malaria mosquitoes. An. gambiae SUA 2La (M) An. gambiae Pimperena (S) An. gambiae Akron (M) An. gambiae Kisumu (S) Pericentric heterochromatin exhibits inter-specific differences An. merus An. quadriannulatus An. arabiensis An. gambiae Zanu (S) Fig. 1. Heterochromatin patterns observed after staining with DAPI. Arrows indicate heterochromatin (darker areas) exhibiting inter and intra-specific differences. While the M forms (Sua, Akron) exhibit single large bands (A, B), S forms (Zanu, Pimperena, Kisumu) exhibit a secondary heterochromatic band in (C-E). Sibling species (FH) also exhibit variation in heterochromatin patterns. An. gambiae Kisumu (S) An. gambiae Akron (M)

Research advance of dosage compensation and MSL complex

Dosage compensation effect, which exists widely in eukaryotes with sexual reproduction, is an essential biological process that equalizes the level of gene expression between genders based on sex determination. In Drosophila, the male-specific lethal (MSL) complex mediates dosage compensation by acetylating histone H4 lysine K16 on nucleosome of some specific sites on the male X chromosome, globally upregulates twofold expression of active X-linked genes from the single X chromosome, and makes up for the shortage that the male has only one single X chromosome in male Drosophila. Up to date, the structure of basic components of MSL complex, which consists of at least five protein subunits and two non-coding RNAs, has already been revealed, and the interaction sites among these components have also been generally identified. Furthermore, abundant researches on recognition mechanism of the complex have been published. In contrast, many studies have revealed that mammalian dosage compensation functions by silencing gene expression from one of the two X chromosomes in females. The main components of mammalian MSL complex have already been identified, but the knowledge of their function is limited. Up to now, research of MSLs in teleosts is scarcely studied. This review summarizes the similarities and differences among dosage compensation mechanisms of nematodes, fruit flies and mammals, introduces the recent research advances in MSL complex, as well as molecular mechanism of dosage compensation in fruit fly, and finally addresses some problems to be resolved. Meanwhile, the diversity of msl3 gene in fishes is found by synteny analysis. This information might provide insightful directions for future research on the mechanisms of dosage compensation in various species.

Re-analysis of the larval testis data on meiotic sex chromosome inactivation revealed evidence for tissue-specific gene expression related to the drosophila X chromosome

BackgroundMeiotic sex chromosome inactivation (MSCI) during spermatogenesis has been proposed as one of the evolutionary driving forces behind both the under-representation of male-biased genes on, and the gene movement out of, the X chromosome in Drosophila. However, the relevance of MSCI in shaping sex chromosome evolution is controversial. Here we examine two aspects of a recent study on testis gene expression (Mikhaylova and Nurminsky, BMC Biol 2011, 9:29) that failed to support the MSCI in Drosophila. First, Mikhaylova and Nurminsky found no differences between X-linked and autosomal genes based on the transcriptional profiling of the early testis development, and thus concluded that MSCI does not occur in D. melanogaster. Second, they also analyzed expression data from several D. melanogaster tissues and concluded that under-representation on the X chromosome is not an exclusive property of testis-biased genes, but instead, a general property of tissue-specific genes.ResultsBy re-analyzing the Mikhaylova and Nurminsky's testis data and the expression data on several D. melanogaster tissues, we made two major findings that refuted their original claims. First, the developmental testis data has generally greater experimental error than conventional analyses, which reduced significantly the power to detect chromosomal differences in expression. Nevertheless, our re-analysis observed significantly lower expression of the X chromosome in the genomic transcriptomes of later development stages of the testis, which is consistent with the MSCI hypothesis. Second, tissue-specific genes are also in general enriched with genes more expressed in testes than in ovaries, that is testis-biased genes. By completely excluding from the analyses the testis-biased genes, which are known to be under-represented in the X, we found that all the other tissue-specific genes are randomly distributed between the X chromosome and the autosomes.ConclusionsOur findings negate the original study of Mikhaylova and Nurminsky, which concluded a lack of MSCI and generalized the pattern of paucity in the X chromosome for tissue-specific genes in Drosophila. Therefore, MSCI and other selection-based models such as sexual antagonism, dosage compensation, and meiotic-drive continue to be viable models as driving forces shaping the genomic distribution of male-related genes in Drosophila.

Autoregulation of the Drosophila Noncoding roX1 RNA Gene

Most genes along the male single X chromosome in Drosophila are hypertranscribed about two-fold relative to each of the two female X chromosomes. This is accomplished by the MSL (male-specific lethal) complex that acetylates histone H4 at lysine 16. The MSL complex contains two large noncoding RNAs, roX1 (RNA on X) and roX2, that help target chromatin modifying enzymes to the X. The roX RNAs are functionally redundant but differ in size, sequence, and transcriptional control. We wanted to find out how roX1 production is regulated. Ectopic DC can be induced in wild-type (roX1+ roX2+) females if we provide a heterologous source of MSL2. However, in the absence of roX2, we found that roX1 expression failed to come on reliably. Using an in situ hybridization probe that is specific only to endogenous roX1, we found that expression was restored if we introduced either roX2 or a truncated but functional version of roX1. This shows that pre-existing roX RNA is required to positively autoregulate roX1 expression. We also observed massive cis spreading of the MSL complex from the site of roX1 transcription at its endogenous location on the X chromosome. We propose that retention of newly assembled MSL complex around the roX gene is needed to drive sustained transcription and that spreading into flanking chromatin contributes to the X chromosome targeting specificity. Finally, we found that the gene encoding the key male-limited protein subunit, msl2, is transcribed predominantly during DNA replication. This suggests that new MSL complex is made as the chromatin template doubles. We offer a model describing how the production of roX1 and msl2, two key components of the MSL complex, are coordinated to meet the dosage compensation demands of the male cell.

Tubby‐RFP balancers for developmental analysis: FM7c 2xTb‐RFP, CyO 2xTb‐RFP, and TM3 2xTb‐RFP

We report here the construction of Tubby-RFP balancers for the X, 2nd and 3rd chromosomes of Drosophila melanogaster. The insertion of a 2xTb-RFP transgene on the FM7c, CyO, and TM3 balancer chromosomes introduces two easily scorable, dominant, developmental markers. The strong Tb phenotype is visible to the naked eye at the larval L2, L3, and pupal stages. The RFP associated with the cuticle is easily detected at all stages from late embryo to adult with the use of a fluorescence stereomicroscope. The FM7c Bar 2xTb-RFP, CyO Cy 2xTb-RFP, and TM3 Sb 2xTb-RFP balancers will greatly facilitate the analysis of lethals and other developmental mutants in L2/L3 larvae and pupae, but also provide coverage of other stages beginning in late embryogenesis through to the adult.

The <i>mus</i>309 mutation, defective in DNA double-strand break repair, increases the frequency of X-ray-induced somatic crossing over in <i>Drosophila melanogaster</i>, but the effect is not dose-rate dependent

Effect of a 1000 R dose of hard X-rays, with two different dose-rates viz. 300 and 1000 R/min on somatic crossing over in the X chromosome of Drosophila melanogaster was studied in two different genotypes. Irradiation was given during the first-instar larval stage of the development. In the control crosses the flies carried wild-type autosomes, but in the experimental crosses the 3rd chromosomes carried a DNA double-strand break repair deficient mus309 mutant gene constitution. As expected, the frequency of X-ray-induced somatic crossing over increased in the mutant flies with both dose-rates of irradiation. As also expected, in the control flies irradiation given with the 300 R/min dose-rate caused more somatic crossovers than irradiation given with the 1000 R/ min rate. However, rather unexpectedly, in the experimental flies there was no significant difference in the frequency of somatic crossing over between the two dose-rates of irradiation. The results can be explained by assuming that X-ray-induced somatic crossing over is a two-step event, and that the mechanism which repairs the lesion caused by the irradiation is controlled by the mus309 gene. In the control flies the repairing mechanism is capable to recover if the irradiation is given with a short term high dose-rate, but is not capable to recover if the irradiation is given with a long lasting low dose-rate. However, in the experimental mutant flies the repairing mechanism is only poorly recovered irrespective of the dose-rate.