Effects of Gene Dose, Chromatin, and Network Topology on Expression in Drosophila melanogaster.

Deletions, commonly referred to as deficiencies by Drosophila geneticists, are valuable tools for mapping genes and for genetic pathway discovery via dose-dependent suppressor and enhancer screens. More recently, it has become clear that deviations from normal gene dosage are associated with multiple disorders in a range of species including humans. While we are beginning to understand some of the transcriptional effects brought about by gene dosage changes and the chromosome rearrangement breakpoints associated with them, much of this work relies on isolated examples. We have systematically examined deficiencies of the left arm of chromosome 2 and characterize gene-by-gene dosage responses that vary from collapsed expression through modest partial dosage compensation to full or even over compensation. We found negligible long-range effects of creating novel chromosome domains at deletion breakpoints, suggesting that cases of gene regulation due to altered nuclear architecture are rare. These rare cases include trans de-repression when deficiencies delete chromatin characterized as repressive in other studies. Generally, effects of breakpoints on expression are promoter proximal (~100bp) or in the gene body. Effects of deficiencies genome-wide are in genes with regulatory relationships to genes within the deleted segments, highlighting the subtle expression network defects in these sensitized genetic backgrounds.

Analysis of the expression patterns, subcellular localisations and interaction partners of Drosophila proteins using a pigP protein trap library.

Although we now have a wealth of information on the transcription patterns of all the genes in the Drosophila genome, much less is known about the properties of the encoded proteins. To provide information on the expression patterns and subcellular localisations of many proteins in parallel, we have performed a large-scale protein trap screen using a hybrid piggyBac vector carrying an artificial exon encoding yellow fluorescent protein (YFP) and protein affinity tags. From screening 41 million embryos, we recovered 616 verified independent YFP-positive lines representing protein traps in 374 genes, two-thirds of which had not been tagged in previous P element protein trap screens. Over 20 different research groups then characterized the expression patterns of the tagged proteins in a variety of tissues and at several developmental stages. In parallel, we purified many of the tagged proteins from embryos using the affinity tags and identified co-purifying proteins by mass spectrometry. The fly stocks are publicly available through the Kyoto Drosophila Genetics Resource Center. All our data are available via an open access database (Flannotator), which provides comprehensive information on the expression patterns, subcellular localisations and in vivo interaction partners of the trapped proteins. Our resource substantially increases the number of available protein traps in Drosophila and identifies new markers for cellular organelles and structures.

How to design a genetic mating scheme: a basic training package for Drosophila genetics.

Drosophila melanogaster is a powerful model organism for biological research. The essential and common instrument of fly research is genetics, the art of applying Mendelian rules in the specific context of Drosophila with its unique classical genetic tools and the breadth of modern genetic tools and strategies brought in by molecular biology, transgenic technologies and the use of recombinases. Training newcomers to fly genetics is a complex and time-consuming task but too important to be left to chance. Surprisingly, suitable training resources for beginners currently are not available. Here we provide a training package for basic Drosophila genetics, designed to ensure that basic knowledge on all key areas is covered while reducing the time invested by trainers. First, a manual introduces to fly history, rationale for mating schemes, fly handling, Mendelian rules in fly, markers and balancers, mating scheme design, and transgenic technologies. Its self-study is followed by a practical training session on gender and marker selection, introducing real flies under the dissecting microscope. Next, through self-study of a PowerPoint presentation, trainees are guided step-by-step through a mating scheme. Finally, to consolidate knowledge, trainees are asked to design similar mating schemes reflecting routine tasks in a fly laboratory. This exercise requires individual feedback but also provides unique opportunities for trainers to spot weaknesses and strengths of each trainee and take remedial action. This training package is being successfully applied at the Manchester fly facility and may serve as a model for further training resources covering other aspects of fly research.

Anent the genomics of spermatogenesis in Drosophila melanogaster.

An appreciable fraction of the Drosophila melanogaster genome is dedicated to male fertility. One approach to characterizing this subset of the genome is through the study of male-sterile mutations. We studied the relation between vital and male-fertility genes in three large autosomal regions that were saturated for lethal and male-sterile mutations. The majority of male-sterile mutations affect genes that are exclusively expressed in males. These genes are required only for male fertility, and several mutant alleles of each such gene were encountered. A few male-sterile mutations were alleles of vital genes that are expressed in both males and females. About one-fifth of the genes in Drosophila melanogaster show male-specific expression in adults. Although some earlier studies found a paucity of genes on the X chromosome showing male-biased expression, we did not find any significant differences between the X chromosome and the autosomes either in the relative frequencies of mutations to male sterility or in the frequencies of genes with male-specific expression in adults. Our results suggest that as much as 25% of the Drosophila genome may be dedicated to male fertility.

Toward a complete Drosophila deficiency kit.

Targeted chromosome engineering has facilitated the development of new deletions that now cover virtually the entire genome of Drosophila, providing greater precision and resolution.

Functional evidence that a recently evolved Drosophila sperm-specific gene boosts sperm competition.

In many species, both morphological and molecular traits related to sex and reproduction evolve faster in males than in females. Ultimately, rapid male evolution relies on the acquisition of genetic variation associated with differential reproductive success. Many newly evolved genes are associated with novel functions that might enhance male fitness. However, functional evidence of the adaptive role of recently originated genes in males is still lacking. The Sperm dynein intermediate chain multigene family, which encodes a Sperm dynein intermediate chain presumably involved in sperm motility, originated from complex genetic rearrangements in the lineage that leads to Drosophila melanogaster within the last 5.4 million years since its split from Drosophila simulans. We deleted all the members of this multigene family resident on the X chromosome of D. melanogaster by chromosome engineering and found that, although the deletion does not result in a reduction of progeny number, it impairs the competence of the sperm in the presence of sperm from wild-type males. Therefore, the Sperm dynein intermediate chain multigene family contributes to the differential reproductive success among males and illustrates precisely how quickly a new gene function can be incorporated into the genetic network of a species.

Modeling serpin conformational diseases in Drosophila melanogaster.

Transgenic Drosophila melanogaster have been used to model both the physiological and pathological behavior of serpins. The ability to generate flies expressing serpins and to rapidly assess associated phenotypes contributes to the power of this paradigm. While providing a whole-organism model of serpinopathies the powerful toolkit of genetic interventions allows precise molecular dissection of important biological pathways. In this chapter, we summarize the contribution that flies have made to the serpin field and then describe some of the experimental methods that are employed in these studies. In particular, we will describe the generation of transgenic flies, the assessment of phenotypes, and the principles of how to perform a genetic screen.

In vivo analysis of proteomes and interactomes using Parallel Affinity Capture (iPAC) coupled to mass spectrometry.

Affinity purification coupled to mass spectrometry provides a reliable method for identifying proteins and their binding partners. In this study we have used Drosophila melanogaster proteins triple tagged with Flag, Strep II, and Yellow fluorescent protein in vivo within affinity pull-down experiments and isolated these proteins in their native complexes from embryos. We describe a pipeline for determining interactomes by Parallel Affinity Capture (iPAC) and show its use by identifying partners of several protein baits with a range of sizes and subcellular locations. This purification protocol employs the different tags in parallel and involves detailed comparison of resulting mass spectrometry data sets, ensuring the interaction lists achieved are of high confidence. We show that this approach identifies known interactors of bait proteins as well as novel interaction partners by comparing data achieved with published interaction data sets. The high confidence in vivo protein data sets presented here add new data to the currently incomplete D. melanogaster interactome. Additionally we report contaminant proteins that are persistent with affinity purifications irrespective of the tagged bait.

Neighbourhood continuity is not required for correct testis gene expression in Drosophila.

It is now widely accepted that gene organisation in eukaryotic genomes is non-random and it is proposed that such organisation may be important for gene expression and genome evolution. In particular, the results of several large-scale gene expression analyses in a range of organisms from yeast to human indicate that sets of genes with similar tissue-specific or temporal expression profiles are clustered within the genome in gene expression neighbourhoods. While the existence of neighbourhoods is clearly established, the underlying reason for this facet of genome organisation is currently unclear and there is little experimental evidence that addresses the genomic requisites for neighbourhood organisation. We report the targeted disruption of three well-defined male-specific gene expression neighbourhoods in the Drosophila genome by the synthesis of precisely mapped chromosomal inversions. We compare gene expression in individuals carrying inverted chromosomes with their non-inverted but otherwise identical progenitors using whole-transcriptome microarray analysis, validating these data with specific quantitative real-time PCR assays. For each neighbourhood we generate and examine multiple inversions. We find no significant differences in the expression of genes that define each of the neighbourhoods. We further show that the inversions spatially separate both halves of a neighbourhood in the nucleus. Thus, models explaining neighbourhood organisation in terms of local sequence interactions, enhancer crosstalk, or short-range chromatin effects are unlikely to account for this facet of genome organisation. Our study challenges the notion that, at least in the case of the testis, expression neighbourhoods are a feature of eukaryotic genome organisation necessary for correct gene expression.

Distinct functions of Mst77F and protamines in nuclear shaping and chromatin condensation during Drosophila spermiogenesis.

Chromatin reorganisation is a major event towards the end of mammalian and Drosophila spermatogenesis. In Drosophila, we previously identified protamine A, protamine B and Mst77F as major chromatin components of the mature sperm. Here, an antibody against Mst77F reveals a dual expression pattern of Mst77F as a chromatin component and in association with microtubules during nuclear shaping. Spermatids of ms(3)nc3 (Mst77F(1)) mutants show disturbed nuclear shaping, instability of perinuclear microtubules but no obvious chromatin condensation defects. Furthermore, we generated a deletion including both protamine genes (prot Delta) and observed that in Drosophila, protamine genes are not haploinsufficient in contrast to those of mice and humans. Moreover, we show that in prot Delta mutants, histone degradation, distribution of DNA breaks and Tpl(94D)-eGFP and Mst77F expression proceed as in wild-type males. Surprisingly, in homozygous prot Delta mutants, males are fertile and sperm are motile, while about 20% of sperm show abnormally shaped nuclei. The latter phenotype can be rescued by supplying protamine-eGFP but not by supplying Mst77F-eGFP. Finally, we demonstrate a 21% increase in X-ray-induced mutation rate of prot Delta sperm. These data support the long-standing hypothesis that the switch from a histone- to protamine-based chromatin protects the paternal genome from mutagens.

The multiple-wing-hairs gene encodes a novel GBD-FH3 domain-containing protein that functions both prior to and after wing hair initiation.

The frizzled signaling/signal transduction pathway controls planar cell polarity (PCP) in both vertebrates and invertebrates. Epistasis experiments argue that in the Drosophila epidermis multiple wing hairs (mwh) acts as a downstream component of the pathway. The PCP proteins accumulate asymmetrically in pupal wing cells where they are thought to form distinct protein complexes. One is located on the distal side of wing cells and a second on the proximal side. This asymmetric protein accumulation is thought to lead to the activation of the cytoskeleton on the distal side, which in turn leads to each cell forming a single distally pointing hair. We identified mwh as CG13913, which encodes a novel G protein binding domain-formin homology 3 (GBD-FH3) domain protein. The Mwh protein accumulated on the proximal side of wing cells prior to hair formation. Unlike planar polarity proteins such as Frizzled or Inturned, Mwh also accumulated in growing hairs. This suggested that mwh had two temporally separate functions in wing development. Evidence for these two functions also came from temperature-shift experiments with a temperature-sensitive allele. Overexpression of Mwh inhibited hair initiation, thus Mwh acts as a negative regulator of the cytoskeleton. Our data argued early proximal Mwh accumulation restricts hair initiation to the distal side of wing cells and the later hair accumulation of Mwh prevents the formation of ectopic secondary hairs. This later function appears to be a feedback mechanism that limits cytoskeleton activation to ensure a single hair is formed.

The ribosomal protein genes and Minute loci of Drosophila melanogaster.

BACKGROUND: Mutations in genes encoding ribosomal proteins (RPs) have been shown to cause an array of cellular and developmental defects in a variety of organisms. In Drosophila melanogaster, disruption of RP genes can result in the 'Minute' syndrome of dominant, haploinsufficient phenotypes, which include prolonged development, short and thin bristles, and poor fertility and viability. While more than 50 Minute loci have been defined genetically, only 15 have so far been characterized molecularly and shown to correspond to RP genes. RESULTS: We combined bioinformatic and genetic approaches to conduct a systematic analysis of the relationship between RP genes and Minute loci. First, we identified 88 genes encoding 79 different cytoplasmic RPs (CRPs) and 75 genes encoding distinct mitochondrial RPs (MRPs). Interestingly, nine CRP genes are present as duplicates and, while all appear to be functional, one member of each gene pair has relatively limited expression. Next, we defined 65 discrete Minute loci by genetic criteria. Of these, 64 correspond to, or very likely correspond to, CRP genes; the single non-CRP-encoding Minute gene encodes a translation initiation factor subunit. Significantly, MRP genes and more than 20 CRP genes do not correspond to Minute loci. CONCLUSION: This work answers a longstanding question about the molecular nature of Minute loci and suggests that Minute phenotypes arise from suboptimal protein synthesis resulting from reduced levels of cytoribosomes. Furthermore, by identifying the majority of haplolethal and haplosterile loci at the molecular level, our data will directly benefit efforts to attain complete deletion coverage of the D. melanogaster genome.

The DrosDel deletion collection: a Drosophila genomewide chromosomal deficiency resource.

We describe a second-generation deficiency kit for Drosophila melanogaster composed of molecularly mapped deletions on an isogenic background, covering approximately 77% of the Release 5.1 genome. Using a previously reported collection of FRT-bearing P-element insertions, we have generated 655 new deletions and verified a set of 209 deletion-bearing fly stocks. In addition to deletions, we demonstrate how the P elements may also be used to generate a set of custom inversions and duplications, particularly useful for balancing difficult regions of the genome carrying haplo-insufficient loci. We describe a simple computational resource that facilitates selection of appropriate elements for generating custom deletions. Finally, we provide a computational resource that facilitates selection of other mapped FRT-bearing elements that, when combined with the DrosDel collection, can theoretically generate over half a million precisely mapped deletions.

Principles of genome evolution in the Drosophila melanogaster species group.

That closely related species often differ by chromosomal inversions was discovered by Sturtevant and Plunkett in 1926. Our knowledge of how these inversions originate is still very limited, although a prevailing view is that they are facilitated by ectopic recombination events between inverted repetitive sequences. The availability of genome sequences of related species now allows us to study in detail the mechanisms that generate interspecific inversions. We have analyzed the breakpoint regions of the 29 inversions that differentiate the chromosomes of Drosophila melanogaster and two closely related species, D. simulans and D. yakuba, and reconstructed the molecular events that underlie their origin. Experimental and computational analysis revealed that the breakpoint regions of 59% of the inversions (17/29) are associated with inverted duplications of genes or other nonrepetitive sequences. In only two cases do we find evidence for inverted repetitive sequences in inversion breakpoints. We propose that the presence of inverted duplications associated with inversion breakpoint regions is the result of staggered breaks, either isochromatid or chromatid, and that this, rather than ectopic exchange between inverted repetitive sequences, is the prevalent mechanism for the generation of inversions in the melanogaster species group. Outgroup analysis also revealed evidence for widespread breakpoint recycling. Lastly, we have found that expression domains in D. melanogaster may be disrupted in D. yakuba, bringing into question their potential adaptive significance.

Culture of Drosophila: the laboratory setup.

INTRODUCTIONSince its introduction to experimental biology more than 90 years ago, Drosophila melanogaster has proved to be an easily cultured and robust laboratory animal. Although culture techniques and the ways in which flies are handled have changed over the years, if he were to enter a fly room today, T.H. Morgan would clearly recognize what is being done, and why. This article provides the basic methods for the laboratory culture of D. melanogaster. The intelligent culture of Drosophila requires a basic understanding of the life cycle of this fly. For this reason, we begin by describing the life cycle of Drosophila and then present information for setting up a fly laboratory.

Maintenance of a Drosophila laboratory: general procedures.

INTRODUCTIONThis article provides a general introduction to keeping Drosophila stocks, making and scoring crosses, mutagenesis, and controlling diseases in the laboratory.

Drosophila crinkled, mutations of which disrupt morphogenesis and cause lethality, encodes fly myosin VIIA.

Myosin VIIs provide motor function for a wide range of eukaryotic processes. We demonstrate that mutations in crinkled (ck) disrupt the Drosophila myosin VIIA heavy chain. The ck/myoVIIA protein is present at a low level throughout fly development and at the same level in heads, thoraxes, and abdomens. Severe ck alleles, likely to be molecular nulls, die as embryos or larvae, but all allelic combinations tested thus far yield a small fraction of adult "escapers" that are weak and infertile. Scanning electron microscopy shows that escapers have defects in bristles and hairs, indicating that this motor protein plays a role in the structure of the actin cytoskeleton. We generate a homology model for the structure of the ck/myosin VIIA head that indicates myosin VIIAs, like myosin IIs, have a spectrin-like, SH3 subdomain fronting their N terminus. In addition, we establish that the two myosin VIIA FERM repeats share high sequence similarity with only the first two subdomains of the three-lobed structure that is typical of canonical FERM domains. Nevertheless, the approximately 100 and approximately 75 amino acids that follow the first two lobes of the first and second FERM domains are highly conserved among myosin VIIs, suggesting that they compose a conserved myosin tail homology 7 (MyTH7) domain that may be an integral part of the FERM domain or may function independently of it. Together, our data suggest a key role for ck/myoVIIA in the formation of cellular projections and other actin-based functions required for viability.

Worniu, a Snail family zinc-finger protein, is required for brain development in Drosophila.

The Snail family of zinc-finger transcriptional repressors is essential for morphogenetic cell movements, mesoderm formation, and neurogenesis during embryonic development. These proteins also control cell cycle, cell death, and cancer progression. In Drosophila, three members of this protein family, Snail, Escargot, and Worniu, have essential but redundant functions in asymmetric cell division of neuroblasts. In addition, Snail is critical for early mesoderm formation and Escargot is required for maintaining diploidy in wing imaginal disc cells. In this report, we demonstrate that Worniu plays a role in brain development. We show that alleles of the l(2)35Da complementation group are mutants of worniu. The developing larvae of these mutant alleles fail to shorten their brainstems. The brain phenotype, as well as the lethality, of these mutants can be rescued by worniu transgenes. Moreover, RNAi experiments targeting the worniu transcript show the same nonshortening phenotype in larval brains. worniu is expressed in the neuroblasts of brain hemispheres and ventral ganglions. The results suggest that the loss of Worniu function within the neuroblasts ultimately causes the larval brainstem to fail to go through shortening during development.

The DrosDel collection: a set of P-element insertions for generating custom chromosomal aberrations in Drosophila melanogaster.

We describe a collection of P-element insertions that have considerable utility for generating custom chromosomal aberrations in Drosophila melanogaster. We have mobilized a pair of engineered P elements, p[RS3] and p[RS5], to collect 3243 lines unambiguously mapped to the Drosophila genome sequence. The collection contains, on average, an element every 35 kb. We demonstrate the utility of the collection for generating custom chromosomal deletions that have their end points mapped, with base-pair resolution, to the genome sequence. The collection was generated in an isogenic strain, thus affording a uniform background for screens where sensitivity to genetic background is high. The entire collection, along with a computational and genetic toolbox for designing and generating custom deletions, is publicly available. Using the collection it is theoretically possible to generate >12,000 deletions between 1 bp and 1 Mb in size by simple eye color selection. In addition, a further 37,000 deletions, selectable by molecular screening, may be generated. We are now using the collection to generate a second-generation deficiency kit that is precisely mapped to the genome sequence.

A new hybrid rescue allele in Drosophila melanogaster.

Crosses of Drosophila melanogaster females to males of its sibling species Drosophila simulans, Drosophila mauritiana and Drosophila sechellia produce no sons and daughters that are viable only at low temperatures. We describe here a novel rescue allele Df(1)EP307-1-2 isolated on the basis of its suppression of high temperature hybrid female lethality. Df(1)EP307-1-2 also rescues hybrid males to the pharate adult stage, the same stage at which it is lethal to D. melanogaster pure species males. Molecular analysis indicates that Df(1)EP307-1-2 is associated with a deletion of about 61 kb in the 9D region of the X chromosome. The structure of Df(1)EP307-1-2 suggests that it was formed by a process similar to P-element induced male recombination.