Surprising Differences in the Variability of Y Chromosomes in African and Cosmopolitan Populations of Drosophila melanogaster

The nonrecombining Drosophila melanogaster Y chromosome is heterochromatic and has few genes. Despite these limitations, there remains ample opportunity for natural selection to act on the genes that are vital for male fertility and on Y factors that modulate gene expression elsewhere in the genome. Y chromosomes of many organisms have low levels of nucleotide variability, but a formal survey of D. melanogaster Y chromosome variation had yet to be performed. Here we surveyed Y-linked variation in six populations of D. melanogaster spread across the globe. We find surprisingly low levels of variability in African relative to Cosmopolitan (i.e., non-African) populations. While the low levels of Cosmopolitan Y chromosome polymorphism can be explained by the demographic histories of these populations, the staggeringly low polymorphism of African Y chromosomes cannot be explained by demographic history. An explanation that is entirely consistent with the data is that the Y chromosomes of Zimbabwe and Uganda populations have experienced recent selective sweeps. Interestingly, the Zimbabwe and Uganda Y chromosomes differ: in Zimbabwe, a European Y chromosome appears to have swept through the population.     

Intercalary heterochromatin in Drosophila melanogaster polytene chromosomes and the problem of genetic silencing

The morphological characteristics of intercalary heterochromatin (IH) are compared with those of other types of silenced chromatin in the Drosophila melanogaster genome: pericentric heterochromatin (PH) and regions subject to position effect variegation (PEV). We conclude that IH regions in polytene chromosomes are binding sites of silencing complexes such as PcG complexes and of SuUR protein. Binding of these proteins results in the appearance of condensed chromatin and late replication of DNA, which in turn may result in DNA underreplication. IH and PH as well as regions subject to PEV have in common the condensed chromatin appearance, the localization of specific proteins, late replication, underreplication in polytene chromosomes, and ectopic pairing.     

Banding patterns in Drosophila melanogaster polytene chromosomes correlate with DNA-binding protein occupancy

The most enigmatic feature of polytene chromosomes is their banding pattern, the genetic organization of which has been a very attractive puzzle for many years. Recent genome-wide protein mapping efforts have produced a wealth of data for the chromosome proteins of Drosophila cells. Based on their specific protein composition, the chromosomes comprise two types of bands, as well as interbands. These differ in terms of time of replication and specific types of proteins. The interbands are characterized by their association with active chromatin proteins, nucleosome remodeling, and origin recognition complexes, and so they have three functions: acting as binding sites for RNA pol II, initiation of replication and nucleosome remodeling of short fragments of DNA. The borders and organization of the same band and interband regions are largely identical, irrespective of the cell type studied. This demonstrates that the banding pattern is a universal principle of the organization of interphase polytene and non-polytene chromosomes.     

Analysis of mutagen specificity Drosophila melanogaster

The previously reported difference between the mutational spectra of hydrazine (HZ) and hydroxylamine (HA) was confirmed for one selected locus (miniature) at which hydrazine produces no mutations in treated late larval spermatogonia or premeiotic spermatocytes sampled by 3 days' progeny. The genetically effective dose was measured in most experiments by the production of v mutants, and in a few by the production of sex-linked lethals. In a total of over 37 000 X-chromosomes (16 000 from previous, and over 21 000 from present experiments) treatment with HZ yielded no m mutation, but 90 v mutations. After treatment with genetically equivalent doses of HA, m and v mutations were about equally frequent. The ratio of visible mutations at the v locus to lethals on the X-chromosomes was exceptionally high after either treatment. So was the ratio of m mutations to lethals after treatment with HA.     

A New Construct for Cloning DNA and Modeling the Structure of Drosophila melanogaster Polytene Chromosomes

Modification of P-element-based transformation vector pCaSpeR3 yielded a new construct, pICon, which contains the structural region of the Escherichia coli lacZ, the adjacent 5 and 3 regulatory regions of hsp70, pUC19, and two tandem FRTs. Owing to the hsp70 promoter, the pICon insertion site may be located on polytene chromosomes after heat shock by light or electron microscopy. The pUC19 sequence with a polylinker allows cloning of the genomic sequence adjacent to the 3 end of pICon by P-target rescue. Functional FRTs allow insertion or deletion of various DNA fragments. The construct is large (22,046 bp), forms easily detectable structures in polytene chromosomes, and may be used to study the structural and functional organization of the Drosophila melanogaster genome, in particular, to elucidate the causes of banding pattern formation. To map the molecular boundaries of interband 3C6/C7, the DNA sequence of this region was cloned between the two FRTs.     

Sisters Unbound Is Required for Meiotic Centromeric Cohesion in Drosophila melanogaster

Regular meiotic chromosome segregation requires sister centromeres to mono-orient (orient to the same pole) during the first meiotic division (meiosis I) when homologous chromosomes segregate, and to bi-orient (orient to opposite poles) during the second meiotic division (meiosis II) when sister chromatids segregate. Both orientation patterns require cohesion between sister centromeres, which is established during meiotic DNA replication and persists until anaphase of meiosis II. Meiotic cohesion is mediated by a conserved four-protein complex called cohesin that includes two structural maintenance of chromosomes (SMC) subunits (SMC1 and SMC3) and two non-SMC subunits. In Drosophila melanogaster, however, the meiotic cohesion apparatus has not been fully characterized and the non-SMC subunits have not been identified. We have identified a novel Drosophila gene called sisters unbound (sunn), which is required for stable sister chromatid cohesion throughout meiosis. sunn mutations disrupt centromere cohesion during prophase I and cause high frequencies of non-disjunction (NDJ) at both meiotic divisions in both sexes. SUNN co-localizes at centromeres with the cohesion proteins SMC1 and SOLO in both sexes and is necessary for the recruitment of both proteins to centromeres. Although SUNN lacks sequence homology to cohesins, bioinformatic analysis indicates that SUNN may be a structural homolog of the non-SMC cohesin subunit stromalin (SA), suggesting that SUNN may serve as a meiosis-specific cohesin subunit. In conclusion, our data show that SUNN is an essential meiosis-specific Drosophila cohesion protein.     

Imaging Through the Pupal Case of Drosophila melanogaster

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.     

Automated identification of social interaction criteria in Drosophila melanogaster

The study of social behaviour within groups has relied on fixed definitions of an ‘interaction’. Criteria used in these definitions often involve a subjectively defined cut-off value for proximity, orientation and time (e.g. courtship, aggression and social interaction networks) and the same numerical values for these criteria are applied to all of the treatment groups within an experiment. One universal definition of an interaction could misidentify interactions within groups that differ in life histories, study treatments and/or genetic mutations. Here, we present an automated method for determining the values of interaction criteria using a pre-defined rule set rather than pre-defined values. We use this approach and show changing social behaviours in different manipulations of Drosophila melanogaster. We also show that chemosensory cues are an important modality of social spacing and interaction. This method will allow a more robust analysis of the properties of interacting groups, while helping us understand how specific groups regulate their social interaction space.     

Genetic analysis of mitochondrial protein misfolding in Drosophila melanogaster

Protein misfolding has a key role in several neurological disorders including Parkinson's disease. Although a clear mechanism for such proteinopathic diseases is well established when aggregated proteins accumulate in the cytosol, cell nucleus, endoplasmic reticulum and extracellular space, little is known about the role of protein aggregation in the mitochondria. Here we show that mutations in both human and fly PINK1 result in higher levels of misfolded components of respiratory complexes and increase in markers of the mitochondrial unfolded protein response. Through the development of a genetic model of mitochondrial protein misfolding employing Drosophila melanogaster, we show that the in vivo accumulation of an unfolded protein in mitochondria results in the activation of AMP-activated protein kinase-dependent autophagy and phenocopies of pink1 and parkin mutants. Parkin expression acts to clear mitochondria with enhanced levels of misfolded proteins by promoting their autophagic degradation in vivo, and refractory to Sigma P (ref(2)P), the Drosophila orthologue of mammalian p62, is a critical downstream effector of this quality control pathway. We show that in flies, a pathway involving pink1, parkin and ref(2)P has a role in the maintenance of a viable pool of cellular mitochondria by promoting organellar quality control.     

Sex-Specific Effects of Cis-Regulatory Variants in Drosophila melanogaster

Sexual dimorphism at the level of gene expression is common and well documented, but much less is known about how different cis-regulatory alleles interact with the different trans-regulatory environments present in males and females. Here we show that sex-specific effects of cis-regulatory variants are common in Drosophila.     

Histone lysine demethylases in Drosophila melanogaster

Epigenetic regulation of chromatin structure is a fundamental process for eukaryotes. Regulators include DNA methylation, microRNAs and chromatin modifications. Within the chromatin modifiers, one class of enzymes that can functionally bind and modify chromatin, through the removal of methyl marks, is the histone lysine demethylases. Here, we summarize the current findings of the 13 known histone lysine demethylases in Drosophila melanogaster, and discuss the critical role of these histone-modifying enzymes in the maintenance of genomic functions. Additionally, as histone demethylase dysregulation has been identified in cancer, we discuss the advantages for using Drosophila as a model system to study tumorigenesis.     

Ribosomal DNA organization before and after magnification in Drosophila melanogaster

In all eukaryotes, the ribosomal RNA genes are stably inherited redundant elements. In Drosophila melanogaster, the presence of a Ybb(-) chromosome in males, or the maternal presence of the Ribosomal exchange (Rex) element, induces magnification: a heritable increase of rDNA copy number. To date, several alternative classes of mechanisms have been proposed for magnification: in situ replication or extra-chromosomal replication, either of which might act on short or extended strings of rDNA units, or unequal sister chromatid exchange. To eliminate some of these hypotheses, none of which has been clearly proven, we examined molecular-variant composition and compared genetic maps of the rDNA in the bb(2) mutant and in some magnified bb(+) alleles. The genetic markers used are molecular-length variants of IGS sequences and of R1 and R2 mobile elements present in many 28S sequences. Direct comparison of PCR products does not reveal any particularly intensified electrophoretic bands in magnified alleles compared to the nonmagnified bb(2) allele. Hence, the increase of rDNA copy number is diluted among multiple variants. We can therefore reject mechanisms of magnification based on multiple rounds of replication of short strings. Moreover, we find no changes of marker order when pre- and postmagnification maps are compared. Thus, we can further restrict the possible mechanisms to two: replication in situ of an extended string of rDNA units or unequal exchange between sister chromatids.     

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.     

Fine-Scale Heterogeneity in Crossover Rate in the garnet-scalloped Region of the Drosophila melanogaster X Chromosome

Homologous recombination affects myriad aspects of genome evolution, from standing levels of nucleotide diversity to the efficacy of natural selection. Rates of crossing over show marked variability at all scales surveyed, including species-, population-, and individual-level differences. Even within genomes, crossovers are nonrandomly distributed in a wide diversity of taxa. Although intra- and intergenomic heterogeneities in crossover distribution have been documented in Drosophila, the scale and degree of crossover rate heterogeneity remain unclear. In addition, the genetic features mediating this heterogeneity are unknown. Here we quantify fine-scale heterogeneity in crossover distribution in a 2.1-Mb region of the Drosophila melanogaster X chromosome by localizing crossover breakpoints in 2500 individuals, each containing a single crossover in this specific X chromosome region. We show 90-fold variation in rates of crossing over at a 5-kb scale, place this variation in the context of several aspects of genome evolution, and identify several genetic features associated with crossover rates. Our results shed new light on the scale and magnitude of crossover rate heterogeneity in D. melanogaster and highlight potential features mediating this heterogeneity.     

How social network structure affects decision-making in Drosophila melanogaster

Animals use a number of different mechanisms to acquire crucial information. During social encounters, animals can pass information from one to another but, ideally, they would only use information that benefits survival and reproduction. Therefore, individuals need to be able to determine the value of the information they receive. One cue can come from the behaviour of other individuals that are already using the information. Using a previous extended dataset, we studied how individual decision-making is influenced by the behaviour of conspecifics in Drosophila melanogaster. We analysed how uninformed flies acquire and later use information about oviposition site choice they learn from informed flies. Our results suggest that uninformed flies adjust their future choices based on how coordinated the behaviours of the informed individuals they encounter are. Following social interaction, uninformed flies tended either to collectively follow the choice of the informed flies or to avoid it. Using social network analysis, we show that this selective information use seems to be based on the level of homogeneity of the social network. In particular, we found that the variance of individual centrality parameters among informed flies was lower in the case of a ‘follow’ outcome compared with the case of an ‘avoid’ outcome.