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.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
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.
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.
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 (
protΔ) and observed that in
Drosophila, protamine genes are not haploinsufficient in contrast to those of mice and humans. Moreover, we show that in
protΔ mutants, histone degradation, distribution of DNA breaks and Tpl
94D-eGFP and Mst77F expression proceed as in wild-type males. Surprisingly, in homozygous
protΔ 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
protΔ sperm. These data support the long-standing hypothesis that the switch from a histone- to protamine-based chromatin protects the paternal genome from mutagens.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Matings among different species of animals or plants often result in sterile or lethal hybrids. Identifying the evolutionary forces that create hybrid incompatibility alleles is fundamental to ...understanding the process of speciation, but very few such alleles have been identified, particularly in model organisms that are amenable to experimental manipulation. We report here the cloning of the first, to our knowledge, Drosophila melanogaster gene involved in hybrid incompatibilities, Hybrid male rescue (Hmr). Hmr causes lethality and female sterility in hybrids among D. melanogaster and its sibling species. We have found that Hmr encodes a protein with homology to a family of MYB-related DNA-binding transcriptional regulators. The HMR protein has evolved both amino acid substitutions and insertions and deletions at an extraordinarily high rate between D. melanogaster and its sibling species, including in its predicted DNA-binding domain. Our results suggest that hybrid lethality may result from disruptions in gene regulation, and we also propose that rapid evolution may be a hallmark of speciation genes in general.
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.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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