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1.  Automated protein-DNA interaction screening of Drosophila regulatory elements 
Nature methods  2011;8(12):1065-1070.
Drosophila melanogaster has one of the best characterized metazoan genomes in terms of functionally annotated regulatory elements. To explore how these elements contribute to gene regulation in the context of gene regulatory networks, we need convenient tools to identify the proteins that bind to them. Here, we present the development and validation of a highly automated protein-DNA interaction detection method, enabling the high-throughput yeast one-hybrid-based screening of DNA elements versus an array of full-length, sequence-verified clones containing 647 (over 85%) of predicted Drosophila transcription factors (TFs). Using six well-characterized regulatory elements (82 bp – 1kb), we identified 33 TF-DNA interactions of which 27 are novel. To simultaneously validate these interactions and locate their binding sites of involved TFs, we implemented a novel microfluidics-based approach that enables us to conduct hundreds of gel shift-like assays at once, thus allowing the retrieval of DNA occupancy data for each TF throughout the respective target DNA elements. Finally, we biologically validate several interactions and specifically identify two novel regulators of sine oculis gene expression and hence eye development.
doi:10.1038/nmeth.1763
PMCID: PMC3929264  PMID: 22037703
2.  An extracellular interactome of Immunoglobulin and LRR proteins reveals receptor-ligand networks 
Cell  2013;154(1):228-239.
Extracellular domains of cell-surface receptors and ligands mediate cell-cell communication, adhesion, and initiation of signaling events, but most existing protein-protein “interactome” datasets lack information for extracellular interactions. We probed interactions between receptor extracellular domains, focusing on the Immunoglobulin Superfamily (IgSF), Fibronectin type-III (FnIII) and Leucine-rich repeat (LRR) families of Drosophila, a set of 202 proteins, many of which are known to be important in neuronal and developmental functions. Out of 20503 candidate protein pairs tested, we observed 106 interactions, 83 of which were previously unknown. We ‘deorphanized’ the 20-member subfamily of defective in proboscis IgSF proteins, showing that they selectively interact with an 11-member subfamily of previously uncharacterized IgSF proteins. Both subfamilies interact with a single common ‘orphan’ LRR protein. We also observed new interactions between Hedgehog and EGFR pathway components. Several of these interactions could be visualized in live-dissected embryos, demonstrating that this approach can identify physiologically relevant receptor-ligand pairs.
doi:10.1016/j.cell.2013.06.006
PMCID: PMC3756661  PMID: 23827685
4.  Computational Identification of Diverse Mechanisms Underlying Transcription Factor-DNA Occupancy 
PLoS Genetics  2013;9(8):e1003571.
ChIP-based genome-wide assays of transcription factor (TF) occupancy have emerged as a powerful, high-throughput method to understand transcriptional regulation, especially on a global scale. This has led to great interest in the underlying biochemical mechanisms that direct TF-DNA binding, with the ultimate goal of computationally predicting a TF's occupancy profile in any cellular condition. In this study, we examined the influence of various potential determinants of TF-DNA binding on a much larger scale than previously undertaken. We used a thermodynamics-based model of TF-DNA binding, called “STAP,” to analyze 45 TF-ChIP data sets from Drosophila embryonic development. We built a cross-validation framework that compares a baseline model, based on the ChIP'ed (“primary”) TF's motif, to more complex models where binding by secondary TFs is hypothesized to influence the primary TF's occupancy. Candidates interacting TFs were chosen based on RNA-SEQ expression data from the time point of the ChIP experiment. We found widespread evidence of both cooperative and antagonistic effects by secondary TFs, and explicitly quantified these effects. We were able to identify multiple classes of interactions, including (1) long-range interactions between primary and secondary motifs (separated by ≤150 bp), suggestive of indirect effects such as chromatin remodeling, (2) short-range interactions with specific inter-site spacing biases, suggestive of direct physical interactions, and (3) overlapping binding sites suggesting competitive binding. Furthermore, by factoring out the previously reported strong correlation between TF occupancy and DNA accessibility, we were able to categorize the effects into those that are likely to be mediated by the secondary TF's effect on local accessibility and those that utilize accessibility-independent mechanisms. Finally, we conducted in vitro pull-down assays to test model-based predictions of short-range cooperative interactions, and found that seven of the eight TF pairs tested physically interact and that some of these interactions mediate cooperative binding to DNA.
Author Summary
Chromatin Immunoprecipitation (ChIP)-based genome-wide assays of transcription factor (TF) occupancy have emerged as a powerful, high throughput method to understand transcriptional regulation, especially on a global scale. Here, we utilize 45 ChIP-chip and ChIP-SEQ data sets from Drosophila to explore the underlying mechanisms of TF-DNA binding. For this, we employ a biophysically motivated computational model, in conjunction with over 300 TF motifs (binding specificities) as well as gene expression and DNA accessibility data from different developmental stages in Drosophila embryos. Our findings provide robust statistical evidence of the role played by TF-TF interactions in shaping genome-wide TF-DNA binding profiles, and thus in directing gene regulation. Our method allows us to go beyond simply recognizing the existence of such interactions, to quantifying their effects on TF occupancy. We are able to categorize the probable mechanisms of these effects as involving direct physical interactions versus accessibility-mediated indirect interactions, long-range versus short-range interactions, and cooperative versus antagonistic interactions. Our analysis reveals widespread evidence of combinatorial regulation present in recently generated ChIP data sets, and sets the stage for rich integrative models of the future that will predict cell type-specific TF occupancy values from sequence and expression data.
doi:10.1371/journal.pgen.1003571
PMCID: PMC3731213  PMID: 23935523
5.  Global Patterns of Tissue-Specific Alternative Polyadenylation in Drosophila 
Cell reports  2012;1(3):277-289.
SUMMARY
We analyzed the usage and consequences of alternative cleavage and polyadenylation (APA) in Drosophila melanogaster by using >1 billion reads of stranded mRNA-seq across a variety of dissected tissues. Beyond demonstrating that a majority of fly transcripts are subject to APA, we observed broad trends for 3′ untranslated region (UTR) shortening in the testis and lengthening in the central nervous system (CNS); the latter included hundreds of unannotated extensions ranging up to 18 kb. Extensive northern analyses validated the accumulation of full-length neural extended transcripts, and in situ hybridization indicated their spatial restriction to the CNS. Genes encoding RNA binding proteins (RBPs) and transcription factors were preferentially subject to 3′ UTR extensions. Motif analysis indicated enrichment of miRNA and RBP sites in the neural extensions, and their termini were enriched in canonical cis elements that promote cleavage and polyadenylation. Altogether, we reveal broad tissue-specific patterns of APA in Drosophila and transcripts with unprecedented 3′ UTR length in the nervous system.
doi:10.1016/j.celrep.2012.01.001
PMCID: PMC3368434  PMID: 22685694
6.  Quantitative Analysis of the Drosophila Segmentation Regulatory Network Using Pattern Generating Potentials 
PLoS Biology  2010;8(8):e1000456.
A new computational method uses gene expression databases and transcription factor binding specificities to describe regulatory elements in the Drosophila A/P patterning network in unprecedented detail.
Cis-regulatory modules that drive precise spatial-temporal patterns of gene expression are central to the process of metazoan development. We describe a new computational strategy to annotate genomic sequences based on their “pattern generating potential” and to produce quantitative descriptions of transcriptional regulatory networks at the level of individual protein-module interactions. We use this approach to convert the qualitative understanding of interactions that regulate Drosophila segmentation into a network model in which a confidence value is associated with each transcription factor-module interaction. Sequence information from multiple Drosophila species is integrated with transcription factor binding specificities to determine conserved binding site frequencies across the genome. These binding site profiles are combined with transcription factor expression information to create a model to predict module activity patterns. This model is used to scan genomic sequences for the potential to generate all or part of the expression pattern of a nearby gene, obtained from available gene expression databases. Interactions between individual transcription factors and modules are inferred by a statistical method to quantify a factor's contribution to the module's pattern generating potential. We use these pattern generating potentials to systematically describe the location and function of known and novel cis-regulatory modules in the segmentation network, identifying many examples of modules predicted to have overlapping expression activities. Surprisingly, conserved transcription factor binding site frequencies were as effective as experimental measurements of occupancy in predicting module expression patterns or factor-module interactions. Thus, unlike previous module prediction methods, this method predicts not only the location of modules but also their spatial activity pattern and the factors that directly determine this pattern. As databases of transcription factor specificities and in vivo gene expression patterns grow, analysis of pattern generating potentials provides a general method to decode transcriptional regulatory sequences and networks.
Author Summary
The developmental program specifying segmentation along the anterior-posterior axis of the Drosophila embryo is one of the best studied examples of transcriptional regulatory networks. Previous work has identified the location and function of dozens of DNA segments called cis-regulatory “modules” that regulate several genes in precise spatial patterns in the early embryo. In many cases, transcription factors that interact with such modules have also been identified. We present a novel computational framework that turns a qualitative and fragmented understanding of modules and factor-module interactions into a quantitative, systems-level view. The formalism utilizes experimentally characterized binding specificities of transcription factors and gene expression patterns to describe how multiple transcription factors (working as activators or repressors) act together in a module to determine its regulatory activity. This formalism can explain the expression patterns of known modules, infer factor-module interactions and quantify the potential of an arbitrary DNA segment to drive a gene's expression. We have also employed databases of gene expression patterns to find novel modules of the regulatory network. As databases of binding motifs and gene expression patterns grow, this new approach provides a general method to decode transcriptional regulatory sequences and networks.
doi:10.1371/journal.pbio.1000456
PMCID: PMC2923081  PMID: 20808951
7.  Unlocking the secrets of the genome 
Nature  2009;459(7249):927-930.
Despite the successes of genomics, little is known about how genetic information produces complex organisms. A look at the crucial functional elements of fly and worm genomes could change that.
doi:10.1038/459927a
PMCID: PMC2843545  PMID: 19536255
8.  Sequence Finishing and Mapping of Drosophila melanogaster Heterochromatin 
Science (New York, N.Y.)  2007;316(5831):1625-1628.
Genome sequences for most metazoans and plants are incomplete because of the presence of repeated DNA in the heterochromatin. The heterochromatic regions of Drosophila melanogaster contain 20 million bases (Mb) of sequence amenable to mapping, sequence assembly, and finishing. We describe the generation of 15 Mb of finished or improved heterochromatic sequence with the use of available clone resources and assembly methods. We also constructed a bacterial artificial chromosome–based physical map that spans 13 Mb of the pericentromeric heterochromatin and a cytogenetic map that positions 11 Mb in specific chromosomal locations. We have approached a complete assembly and mapping of the nonsatellite component of Drosophila heterochromatin. The strategy we describe is also applicable to generating substantially more information about heterochromatin in other species, including humans.
doi:10.1126/science.1139816
PMCID: PMC2825053  PMID: 17569867
9.  Systematic image-driven analysis of the spatial Drosophila embryonic expression landscape 
We created innovative virtual representation for our large scale Drosophila insitu expression dataset. We aligned an elliptically shaped mesh comprised of small triangular regions to the outline of each embryo. Each triangle defines a unique location in the embryo and comparing corresponding triangles allows easy identification of similar expression patterns.The virtual representation was used to organize the expression landscape at stage 4-6. We identified regions with similar expression in the embryo and clustered genes with similar expression patterns.We created algorithms to mine the dataset for adjacent non-overlapping patterns and anti-correlated patterns. We were able to mine the dataset to identify co-expressed and putative interacting genes.Using co-expression we were able to assign putative functions to unknown genes.
Analyzing both temporal and spatial gene expression is essential for understanding development and regulatory networks of multicellular organisms. Interacting genes are commonly expressed in overlapping or adjacent domains. Thus, gene expression patterns can be used to assign putative gene functions and mined to infer candidates for networks.
We have generated a systematic two-dimensional mRNA expression atlas profiling embryonic development of Drosophila melanogaster (Tomancak et al, 2002, 2007). To date, we have collected over 70 000 images for over 6000 genes. To explore spatial relationships between gene expression patterns, we used a novel computational image-processing approach by converting expression patterns from the images into virtual representations (Figure 1). Using a custom-designed automated pipeline, for each image, we segmented and aligned the outline of the embryo to an elliptically shaped mesh, comprised of 311 small triangular regions each defining a unique location within the embryo. By comparing corresponding triangles, we produced a distance score to identify similar patterns. We generated those triangulated images (TIs) for our entire data set at all developmental stages and demonstrated that this representation can be used as for objective computationally defined description for expression in in situ hybridization images from various sources, including images from the literature.
We used the TIs to conduct a comprehensive analysis of the expression landscape. To this end, we created a novel approach to temporally sort and compact TIs to a non-redundant data set suitable for further computational processing. Although generally applicable for all developmental stages, for this study, we focused on developmental stages 4–6. For this stage range, we reduced the initial set of about 5800 TIs to 553 TIs containing 364 genes. Using this filtered data set, to discover how expression subdivides the embryo into regions, we clustered areas with similar expression and demonstrated that expression patterns divide the early embryo into distinct spatial regions resembling a fate map (Figure 3). To discover the range of unique expression patterns, we used affinity propagation clustering (Frey and Dueck, 2007) to group TIs with similar patterns and identified 39 clusters each representing a distinct pattern class. We integrated the remaining genes into the 39 clusters and studied the distribution of expression patterns and the relationships between the clusters.
The clustered expression patterns were used to identify putative positive and negative regulatory interactions. The similar TIs in each cluster not only grouped already known genes with related functions, but previously undescribed genes. A comparative analysis identified subtle differences between the genes within each expression cluster. To investigate these differences, we developed a novel Markov Random Field (MRF) segmentation algorithm to extract patterns. We then extended the MRF algorithm to detect shared expression boundaries, generate similarity measurements, and discriminate even faint/uncertain patterns between two TIs. This enabled us to identify more subtle partial expression pattern overlaps and adjacent non-overlapping patterns. For example, by conducting this analysis on the cluster containing the gene snail, we identified the previously known huckebein, which restricts snail expression (Reuter and Leptin, 1994), and zfh1, which interacts with tinman (Broihier et al, 1998; Su et al, 1999).
By studying the functions of known genes, we assigned putative developmental roles to each of the 39 clusters. Of the 1800 genes investigated, only half of them had previously assigned functions.
Representing expression patterns with geometric meshes facilitates the analysis of a complex process involving thousands of genes. This approach is complementary to the cellular resolution 3D atlas for the Drosophila embryo (Fowlkes et al, 2008). Our method can be used as a rapid, fully automated, high-throughput approach to obtain a map of co-expression, which will serve to select specific genes for detailed multiplex in-situ hybridization and confocal analysis for a fine-grain atlas. Our data are similar to the data in the literature, and research groups studying reporter constructs, mutant animals, or orthologs can easily produce in situ hybridizations. TIs can be readily created and provide representations that are both comparable to each other and our data set. We have demonstrated that our approach can be used for predicting relationships in regulatory and developmental pathways.
Discovery of temporal and spatial patterns of gene expression is essential for understanding the regulatory networks and development in multicellular organisms. We analyzed the images from our large-scale spatial expression data set of early Drosophila embryonic development and present a comprehensive computational image analysis of the expression landscape. For this study, we created an innovative virtual representation of embryonic expression patterns using an elliptically shaped mesh grid that allows us to make quantitative comparisons of gene expression using a common frame of reference. Demonstrating the power of our approach, we used gene co-expression to identify distinct expression domains in the early embryo; the result is surprisingly similar to the fate map determined using laser ablation. We also used a clustering strategy to find genes with similar patterns and developed new analysis tools to detect variation within consensus patterns, adjacent non-overlapping patterns, and anti-correlated patterns. Of the 1800 genes investigated, only half had previously assigned functions. The known genes suggest developmental roles for the clusters, and identification of related patterns predicts requirements for co-occurring biological functions.
doi:10.1038/msb.2009.102
PMCID: PMC2824522  PMID: 20087342
biological function; embryo; gene expression; in situ hybridization; Markov Random Field
10.  Determination of gene expression patterns using high-throughput RNA in situ hybridization to whole-mount Drosophila embryos 
Nature protocols  2009;4(5):605-618.
We describe a high-throughput protocol for RNA in situ hybridization (ISH) to Drosophila embryos in 96-well format. cDNA or genomic DNA templates are amplified by PCR and then digoxigenin-labeled ribonucleotides are incorporated into anti-sense RNA probes by in vitro transcription. The quality of each probe is evaluated prior to in situ hybridization using a RNA Probe Quantification (dot blot) assay. RNA probes are hybridized to fixed, mixed-staged Drosophila embryos in 96-well plates. The resulting stained embryos can be examined and photographed immediately or stored at 4°C for later analysis. Starting with fixed, staged embryos, the protocol takes 6 days from probe template production through hybridization. Preparation of fixed embryos requires a minimum of two weeks to collect embryos representing all stages. The method has been used to determine the expression patterns of over 6000 genes throughout embryogenesis.
doi:10.1038/nprot.2009.55
PMCID: PMC2780369  PMID: 19360017
11.  Comparative Genomics of the Eukaryotes 
Science (New York, N.Y.)  2000;287(5461):2204-2215.
A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae—and the proteins they are predicted to encode—was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.
PMCID: PMC2754258  PMID: 10731134
12.  Regulation of Early Endosomal Entry by the Drosophila Tumor Suppressors Rabenosyn and Vps45 
Molecular Biology of the Cell  2008;19(10):4167-4176.
The small GTPase Rab5 has emerged as an important regulator of animal development, and it is essential for endocytic trafficking. However, the mechanisms that link Rab5 activation to cargo entry into early endosomes remain unclear. We show here that Drosophila Rabenosyn (Rbsn) is a Rab5 effector that bridges an interaction between Rab5 and the Sec1/Munc18-family protein Vps45, and we further identify the syntaxin Avalanche (Avl) as a target for Vps45 activity. Rbsn and Vps45, like Avl and Rab5, are specifically localized to early endosomes and are required for endocytosis. Ultrastructural analysis of rbsn, Vps45, avl, and Rab5 null mutant cells, which show identical defects, demonstrates that all four proteins are required for vesicle fusion to form early endosomes. These defects lead to loss of epithelial polarity in mutant tissues, which overproliferate to form neoplastic tumors. This work represents the first characterization of a Rab5 effector as a tumor suppressor, and it provides in vivo evidence for a Rbsn–Vps45 complex on early endosomes that links Rab5 to the SNARE fusion machinery.
doi:10.1091/mbc.E08-07-0716
PMCID: PMC2555928  PMID: 18685079
14.  Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes 
Nature genetics  2008;40(4):476-483.
A major obstacle to creating precisely expressed transgenes lies in the epigenetic effects of the host chromatin that surrounds them. Here we present a strategy to overcome this problem, employing a Gal4-inducible luciferase assay to systematically quantify position effects of host chromatin and the ability of insulators to counteract these effects at phiC31 integration loci randomly distributed throughout the Drosophila genome. We identify loci that can be exploited to deliver precise doses of transgene expression to specific tissues. Moreover, we uncover a previously unrecognized property of the gypsy retrovirus insulator to boost gene expression to levels severalfold greater than at most or possibly all un-insulated loci, in every tissue tested. These findings provide the first opportunity to create a battery of transgenes that can be reliably expressed at high levels in virtually any tissue by integration at a single locus, and conversely, to engineer a controlled phenotypic allelic series by exploiting several loci. The generality of our approach makes it adaptable to other model systems to identify and modify loci for optimal transgene expression.
doi:10.1038/ng.101
PMCID: PMC2330261  PMID: 18311141
15.  Improved repeat identification and masking in Dipterans 
Gene  2006;389(1):1-9.
doi:10.1016/j.gene.2006.09.011
PMCID: PMC1945102  PMID: 17137733
Heterochromatin; Drosophila; A. gambiae; PILER; transposable element; RepeatRunner
16.  Global analysis of patterns of gene expression during Drosophila embryogenesis 
Genome Biology  2007;8(7):R145.
Embryonic expression patterns for 6,003 (44%) of the 13,659 protein-coding genes identified in the Drosophila melanogaster genome were documented, of which 40% show tissue-restricted expression.
Background
Cell and tissue specific gene expression is a defining feature of embryonic development in multi-cellular organisms. However, the range of gene expression patterns, the extent of the correlation of expression with function, and the classes of genes whose spatial expression are tightly regulated have been unclear due to the lack of an unbiased, genome-wide survey of gene expression patterns.
Results
We determined and documented embryonic expression patterns for 6,003 (44%) of the 13,659 protein-coding genes identified in the Drosophila melanogaster genome with over 70,000 images and controlled vocabulary annotations. Individual expression patterns are extraordinarily diverse, but by supplementing qualitative in situ hybridization data with quantitative microarray time-course data using a hybrid clustering strategy, we identify groups of genes with similar expression. Of 4,496 genes with detectable expression in the embryo, 2,549 (57%) fall into 10 clusters representing broad expression patterns. The remaining 1,947 (43%) genes fall into 29 clusters representing restricted expression, 20% patterned as early as blastoderm, with the majority restricted to differentiated cell types, such as epithelia, nervous system, or muscle. We investigate the relationship between expression clusters and known molecular and cellular-physiological functions.
Conclusion
Nearly 60% of the genes with detectable expression exhibit broad patterns reflecting quantitative rather than qualitative differences between tissues. The other 40% show tissue-restricted expression; the expression patterns of over 1,500 of these genes are documented here for the first time. Within each of these categories, we identified clusters of genes associated with particular cellular and developmental functions.
doi:10.1186/gb-2007-8-7-r145
PMCID: PMC2323238  PMID: 17645804
17.  Drosophila by the dozen 
Genome Biology  2007;8(7):309.
A report of the 48th Annual Drosophila Research Conference, Philadelphia, USA, 7-11 March 2007.
A report of the 48th Annual Drosophila Research Conference, Philadelphia, USA, 7-11 March 2007.
doi:10.1186/gb-2007-8-7-309
PMCID: PMC2323228  PMID: 17634150
18.  Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP) 
Nucleic Acids Research  2005;33(21):e185.
cDNA cloning is a central technology in molecular biology. cDNA sequences are used to determine mRNA transcript structures, including splice junctions, open reading frames (ORFs) and 5′- and 3′-untranslated regions (UTRs). cDNA clones are valuable reagents for functional studies of genes and proteins. Expressed Sequence Tag (EST) sequencing is the method of choice for recovering cDNAs representing many of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a cDNA library at random, and it recovers transcripts with low expression levels inefficiently. We describe a PCR-based method for directed screening of plasmid cDNA libraries. We demonstrate its utility in a screen of libraries used in our Drosophila EST projects for 153 transcription factor genes that were not represented by full-length cDNA clones in our Drosophila Gene Collection. We recovered high-quality, full-length cDNAs for 72 genes and variously compromised clones for an additional 32 genes. The method can be used at any scale, from the isolation of cDNA clones for a particular gene of interest, to the improvement of large gene collections in model organisms and the human. Finally, we discuss the relative merits of directed cDNA library screening and RT–PCR approaches.
doi:10.1093/nar/gni184
PMCID: PMC1301602  PMID: 16326860
19.  Benchmarking tools for the alignment of functional noncoding DNA 
BMC Bioinformatics  2004;5:6.
Background
Numerous tools have been developed to align genomic sequences. However, their relative performance in specific applications remains poorly characterized. Alignments of protein-coding sequences typically have been benchmarked against "correct" alignments inferred from structural data. For noncoding sequences, where such independent validation is lacking, simulation provides an effective means to generate "correct" alignments with which to benchmark alignment tools.
Results
Using rates of noncoding sequence evolution estimated from the genus Drosophila, we simulated alignments over a range of divergence times under varying models incorporating point substitution, insertion/deletion events, and short blocks of constrained sequences such as those found in cis-regulatory regions. We then compared "correct" alignments generated by a modified version of the ROSE simulation platform to alignments of the simulated derived sequences produced by eight pairwise alignment tools (Avid, BlastZ, Chaos, ClustalW, DiAlign, Lagan, Needle, and WABA) to determine the off-the-shelf performance of each tool. As expected, the ability to align noncoding sequences accurately decreases with increasing divergence for all tools, and declines faster in the presence of insertion/deletion evolution. Global alignment tools (Avid, ClustalW, Lagan, and Needle) typically have higher sensitivity over entire noncoding sequences as well as in constrained sequences. Local tools (BlastZ, Chaos, and WABA) have lower overall sensitivity as a consequence of incomplete coverage, but have high specificity to detect constrained sequences as well as high sensitivity within the subset of sequences they align. Tools such as DiAlign, which generate both local and global outputs, produce alignments of constrained sequences with both high sensitivity and specificity for divergence distances in the range of 1.25–3.0 substitutions per site.
Conclusion
For species with genomic properties similar to Drosophila, we conclude that a single pair of optimally diverged species analyzed with a high performance alignment tool can yield accurate and specific alignments of functionally constrained noncoding sequences. Further algorithm development, optimization of alignment parameters, and benchmarking studies will be necessary to extract the maximal biological information from alignments of functional noncoding DNA.
doi:10.1186/1471-2105-5-6
PMCID: PMC344529  PMID: 14736341
20.  Heterochromatic sequences in a Drosophila whole-genome shotgun assembly 
Genome Biology  2002;3(12):research0085.1-85.16.
Annotation of an improved whole-genome shotgun assembly of the Drosophila melanogaster genome predicted 297 protein-coding genes and six non-protein-coding genes, including known heterochromatic genes, and regions of similarity to known transposable elements. Fluorescence in situ hybridization was used to correlate the genomic sequence with the cytogenetic map; the annotated euchromatic sequence extends into the centric heterochromatin on each chromosome arm.
Background
Most eukaryotic genomes include a substantial repeat-rich fraction termed heterochromatin, which is concentrated in centric and telomeric regions. The repetitive nature of heterochromatic sequence makes it difficult to assemble and analyze. To better understand the heterochromatic component of the Drosophila melanogaster genome, we characterized and annotated portions of a whole-genome shotgun sequence assembly.
Results
WGS3, an improved whole-genome shotgun assembly, includes 20.7 Mb of draft-quality sequence not represented in the Release 3 sequence spanning the euchromatin. We annotated this sequence using the methods employed in the re-annotation of the Release 3 euchromatic sequence. This analysis predicted 297 protein-coding genes and six non-protein-coding genes, including known heterochromatic genes, and regions of similarity to known transposable elements. Bacterial artificial chromosome (BAC)-based fluorescence in situ hybridization analysis was used to correlate the genomic sequence with the cytogenetic map in order to refine the genomic definition of the centric heterochromatin; on the basis of our cytological definition, the annotated Release 3 euchromatic sequence extends into the centric heterochromatin on each chromosome arm.
Conclusions
Whole-genome shotgun assembly produced a reliable draft-quality sequence of a significant part of the Drosophila heterochromatin. Annotation of this sequence defined the intron-exon structures of 30 known protein-coding genes and 267 protein-coding gene models. The cytogenetic mapping suggests that an additional 150 predicted genes are located in heterochromatin at the base of the Release 3 euchromatic sequence. Our analysis suggests strategies for improving the sequence and annotation of the heterochromatic portions of the Drosophila and other complex genomes.
doi:10.1186/gb-2002-3-12-research0085
PMCID: PMC151187  PMID: 12537574
21.  Systematic determination of patterns of gene expression during Drosophila embryogenesis 
Genome Biology  2002;3(12):research0088.1-88.14.
As a first step to creating a comprehensive atlas of gene-expression patterns during Drosophila embryogenesis, 2,179 genes have been examinded by in situ hybridization to fixed Drosophila embryos. Of the genes assayed, 63.7% displayed dynamic expression patterns that were documented with 25,690 digital photomicrographs of individual embryos.
Background
Cell-fate specification and tissue differentiation during development are largely achieved by the regulation of gene transcription.
Results
As a first step to creating a comprehensive atlas of gene-expression patterns during Drosophila embryogenesis, we examined 2,179 genes by in situ hybridization to fixed Drosophila embryos. Of the genes assayed, 63.7% displayed dynamic expression patterns that were documented with 25,690 digital photomicrographs of individual embryos. The photomicrographs were annotated using controlled vocabularies for anatomical structures that are organized into a developmental hierarchy. We also generated a detailed time course of gene expression during embryogenesis using microarrays to provide an independent corroboration of the in situ hybridization results. All image, annotation and microarray data are stored in publicly available database. We found that the RNA transcripts of about 1% of genes show clear subcellular localization. Nearly all the annotated expression patterns are distinct. We present an approach for organizing the data by hierarchical clustering of annotation terms that allows us to group tissues that express similar sets of genes as well as genes displaying similar expression patterns.
Conclusions
Analyzing gene-expression patterns by in situ hybridization to whole-mount embryos provides an extremely rich dataset that can be used to identify genes involved in developmental processes that have been missed by traditional genetic analysis. Systematic analysis of rigorously annotated patterns of gene expression will complement and extend the types of analyses carried out using expression microarrays.
doi:10.1186/gb-2002-3-12-research0088
PMCID: PMC151190  PMID: 12537577
22.  The transposable elements of the Drosophila melanogaster euchromatin: a genomics perspective 
Genome Biology  2002;3(12):research0084.1-84.2.
Using Release 3 of the euchromatic genomic sequence of Drosophila melanogaster, 85 known and eight novel families of transposable element have been identified, varying in copy number from one to 146. A total of 1,572 full and partial transposable elements were identified, comprising 3.86% of the sequence.
Background
Transposable elements are found in the genomes of nearly all eukaryotes. The recent completion of the Release 3 euchromatic genomic sequence of Drosophila melanogaster by the Berkeley Drosophila Genome Project has provided precise sequence for the repetitive elements in the Drosophila euchromatin. We have used this genomic sequence to describe the euchromatic transposable elements in the sequenced strain of this species.
Results
We identified 85 known and eight novel families of transposable element varying in copy number from one to 146. A total of 1,572 full and partial transposable elements were identified, comprising 3.86% of the sequence. More than two-thirds of the transposable elements are partial. The density of transposable elements increases an average of 4.7 times in the centromere-proximal regions of each of the major chromosome arms. We found that transposable elements are preferentially found outside genes; only 436 of 1,572 transposable elements are contained within the 61.4 Mb of sequence that is annotated as being transcribed. A large proportion of transposable elements is found nested within other elements of the same or different classes. Lastly, an analysis of structural variation from different families reveals distinct patterns of deletion for elements belonging to different classes.
Conclusions
This analysis represents an initial characterization of the transposable elements in the Release 3 euchromatic genomic sequence of D. melanogaster for which comparison to the transposable elements of other organisms can begin to be made. These data have been made available on the Berkeley Drosophila Genome Project website for future analyses.
doi:10.1186/gb-2002-3-12-research0084
PMCID: PMC151186  PMID: 12537573
23.  A Drosophila full-length cDNA resource 
Genome Biology  2002;3(12):research0080.1-80.8.
High-quality full-insert sequence for 8,921 putative full-length cDNA clones in the Drosophila Gene Collection has been generated and compared to the annotated Release 3 genomic sequence. More than 5,300 cDNAs have been identifieed that contain a complete and accurate protein-coding sequence, corresponding to at least one splice form for 40% of the predicted D. melanogaster genes.
Background
A collection of sequenced full-length cDNAs is an important resource both for functional genomics studies and for the determination of the intron-exon structure of genes. Providing this resource to the Drosophila melanogaster research community has been a long-term goal of the Berkeley Drosophila Genome Project. We have previously described the Drosophila Gene Collection (DGC), a set of putative full-length cDNAs that was produced by generating and analyzing over 250,000 expressed sequence tags (ESTs) derived from a variety of tissues and developmental stages.
Results
We have generated high-quality full-insert sequence for 8,921 clones in the DGC. We compared the sequence of these clones to the annotated Release 3 genomic sequence, and identified more than 5,300 cDNAs that contain a complete and accurate protein-coding sequence. This corresponds to at least one splice form for 40% of the predicted D. melanogaster genes. We also identified potential new cases of RNA editing.
Conclusions
We show that comparison of cDNA sequences to a high-quality annotated genomic sequence is an effective approach to identifying and eliminating defective clones from a cDNA collection and ensure its utility for experimentation. Clones were eliminated either because they carry single nucleotide discrepancies, which most probably result from reverse transcriptase errors, or because they are truncated and contain only part of the protein-coding sequence.
doi:10.1186/gb-2002-3-12-research0080
PMCID: PMC151182  PMID: 12537569
24.  Finishing a whole-genome shotgun: Release 3 of the Drosophila melanogaster euchromatic genome sequence 
Genome Biology  2002;3(12):research0079.1-79.14.
The Drosophila melanogaster genome was the first metazoan genome to be sequenced by whole-genome shotgun. Now, the sequence has been finished in a process designed to close gaps, improve sequence quality and validate the assembly.
Background
The Drosophila melanogaster genome was the first metazoan genome to have been sequenced by the whole-genome shotgun (WGS) method. Two issues relating to this achievement were widely debated in the genomics community: how correct is the sequence with respect to base-pair (bp) accuracy and frequency of assembly errors? And, how difficult is it to bring a WGS sequence to the accepted standard for finished sequence? We are now in a position to answer these questions.
Results
Our finishing process was designed to close gaps, improve sequence quality and validate the assembly. Sequence traces derived from the WGS and draft sequencing of individual bacterial artificial chromosomes (BACs) were assembled into BAC-sized segments. These segments were brought to high quality, and then joined to constitute the sequence of each chromosome arm. Overall assembly was verified by comparison to a physical map of fingerprinted BAC clones. In the current version of the 116.9 Mb euchromatic genome, called Release 3, the six euchromatic chromosome arms are represented by 13 scaffolds with a total of 37 sequence gaps. We compared Release 3 to Release 2; in autosomal regions of unique sequence, the error rate of Release 2 was one in 20,000 bp.
Conclusions
The WGS strategy can efficiently produce a high-quality sequence of a metazoan genome while generating the reagents required for sequence finishing. However, the initial method of repeat assembly was flawed. The sequence we report here, Release 3, is a reliable resource for molecular genetic experimentation and computational analysis.
doi:10.1186/gb-2002-3-12-research0079
PMCID: PMC151181  PMID: 12537568
25.  Dynamic reprogramming of chromatin accessibility during Drosophila embryo development 
Genome Biology  2011;12(5):R43.
Background
The development of complex organisms is believed to involve progressive restrictions in cellular fate. Understanding the scope and features of chromatin dynamics during embryogenesis, and identifying regulatory elements important for directing developmental processes remain key goals of developmental biology.
Results
We used in vivo DNaseI sensitivity to map the locations of regulatory elements, and explore the changing chromatin landscape during the first 11 hours of Drosophila embryonic development. We identified thousands of conserved, developmentally dynamic, distal DNaseI hypersensitive sites associated with spatial and temporal expression patterning of linked genes and with large regions of chromatin plasticity. We observed a nearly uniform balance between developmentally up- and down-regulated DNaseI hypersensitive sites. Analysis of promoter chromatin architecture revealed a novel role for classical core promoter sequence elements in directing temporally regulated chromatin remodeling. Another unexpected feature of the chromatin landscape was the presence of localized accessibility over many protein-coding regions, subsets of which were developmentally regulated or associated with the transcription of genes with prominent maternal RNA contributions in the blastoderm.
Conclusions
Our results provide a global view of the rich and dynamic chromatin landscape of early animal development, as well as novel insights into the organization of developmentally regulated chromatin features.
doi:10.1186/gb-2011-12-5-r43
PMCID: PMC3219966  PMID: 21569360

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