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Versatile P(acman) BAC Libraries for Transgenesis Studies in Drosophila melanogaster

Description: We constructed Drosophila melanogaster BAC libraries with 21-kb and 83-kb inserts in the P(acman) system. Clones representing 12-fold coverage and encompassing more than 95percent of annotated genes were mapped onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using Phi C31 integrase to rescue mutations. They can be modified through recombineering, for example to incorporate protein tags and assess expression patterns.
Date: April 21, 2009
Creator: Venken, Koen J.T.; Carlson, Joseph W.; Schulze, Karen L.; Pan, Hongling; He, Yuchun; Spokony, Rebecca et al.
Partner: UNT Libraries Government Documents Department

Drosophila by the dozen

Description: This year's conference on Drosophila research illustratedwell the current focus of Drosophila genomics on the comprehensiveidentification of functional elements in the genome sequence, includingmRNA transcripts arising from multiple alternative start sites and splicesites, a multiplicity of noncoding transcripts and small RNAs,identification of binding sites for transcription factors, sequenceconservation in related species and sequence variation within species.Resources and technologies for genetics and functional genomics aresteadily being improved, including the building of collections oftransposon insertion mutants and hairpin constructs for RNA interference(RNAi). The conference also highlighted progress in the use of genomicinformation by many laboratories to study diverse aspects of biology andmodels of human disease. Here we will review a few highlights of especialinterest to readers of Genome Biology.
Date: July 13, 2007
Creator: Celniker, Susan E. & Hoskins, Roger A.
Partner: UNT Libraries Government Documents Department

Sequence finishing and mapping of Drosophila melanogasterheterochromatin

Description: Genome sequences for most metazoans are incomplete due tothe presence of repeated DNA in the pericentromeric heterochromatin. Theheterochromatic regions of D. melanogaster contain 20 Mb of sequenceamenable to mapping, sequence assembly and finishing. Here we describethe generation of 15 Mb of finished or improved heterochromatic sequenceusing available clone resources and assembly and mapping methods. We alsoconstructed a BAC-based physical map that spans approximately 13 Mb ofthe pericentromeric heterochromatin, and a cytogenetic map that positionsapproximately 11 Mb of BAC contigs and sequence scaffolds in specificchromosomal locations. The integrated sequence assembly and maps greatlyimprove our understanding of the structure and composition of this poorlyunderstood fraction of a metazoan genome and provide a framework forfunctional analyses.
Date: June 15, 2007
Creator: Hoskins, Roger A.; Carlson, Joseph W.; Kennedy, Cameron; Acevedo,David; Evans-Holm, Martha; Frise, Erwin et al.
Partner: UNT Libraries Government Documents Department

PointCloudXplore: a visualization tool for 3D gene expressiondata

Description: The Berkeley Drosophila Transcription Network Project (BDTNP) has developed a suite of methods that support quantitative, computational analysis of three-dimensional (3D) gene expression patterns with cellular resolution in early Drosophila embryos, aiming at a more in-depth understanding of gene regulatory networks. We describe a new tool, called PointCloudXplore (PCX), that supports effective 3D gene expression data exploration. PCX is a visualization tool that uses the established visualization techniques of multiple views, brushing, and linking to support the analysis of high-dimensional datasets that describe many genes' expression. Each of the views in PointCloudXplore shows a different gene expression data property. Brushing is used to select and emphasize data associated with defined subsets of embryo cells within a view. Linking is used to show in additional views the expression data for a group of cells that have first been highlighted as a brush in a single view, allowing further data subset properties to be determined. In PCX, physical views of the data are linked to abstract data displays such as parallel coordinates. Physical views show the spatial relationships between different genes' expression patterns within an embryo. Abstract gene expression data displays on the other hand allow for an analysis of relationships between different genes directly in the gene expression space. We discuss on parallel coordinates as one example abstract data view currently available in PCX. We have developed several extensions to standard parallel coordinates to facilitate brushing and the visualization of 3D gene expression data.
Date: October 1, 2006
Creator: Rubel, Oliver; Weber, Gunther H.; Keranen, Soile V.E.; Fowlkes,Charles C.; Luengo Hendriks, Cristian L.; Simirenko, Lisa et al.
Partner: UNT Libraries Government Documents Department


Description: Drosophila melanogaster adults are irradiated and then mated to flies containing genetic markers and/or crossover suppressors in all chromosomes. Appropriate genetic schemes are followed to obtain balanced stocks of entire treated genomes. Each chromosome of each genome is observed for visibles, lethals, detrimentals and sterility factors. Tests are made for translocations involving any combination of chromosomes. All chromosomes of each genome are examined cytologically for possible deficiencies, inversions, transpositions, etc. and to determine the break points of the translocations. It is believed that a study such as this will give us a far better idea of the overall magnitude of radiation-induced genetic damage and a better insight into the nature of this damage, expecially the interĀ­-relationships between the different kinds of genetic alterations, than has heretofore been possible with studies of selected types of damage in selected chromosomes or loci.
Date: July 28, 1968
Creator: Abrahamson, Seymour
Partner: UNT Libraries Government Documents Department

A BAC-based physical map of the Drosophila buzzatii genome

Description: Large-insert genomic libraries facilitate cloning of large genomic regions, allow the construction of clone-based physical maps and provide useful resources for sequencing entire genomes. Drosophilabuzzatii is a representative species of the repleta group in the Drosophila subgenus, which is being widely used as a model in studies of genome evolution, ecological adaptation and speciation. We constructed a Bacterial Artificial Chromosome (BAC) genomic library of D. buzzatii using the shuttle vector pTARBAC2.1. The library comprises 18,353 clones with an average insert size of 152 kb and a {approx}18X expected representation of the D. buzzatii euchromatic genome. We screened the entire library with six euchromatic gene probes and estimated the actual genome representation to be {approx}23X. In addition, we fingerprinted by restriction digestion and agarose gel electrophoresis a sample of 9,555 clones, and assembled them using Finger Printed Contigs (FPC) software and manual editing into 345 contigs (mean of 26 clones per contig) and 670singletons. Finally, we anchored 181 large contigs (containing 7,788clones) to the D. buzzatii salivary gland polytene chromosomes by in situ hybridization of 427 representative clones. The BAC library and a database with all the information regarding the high coverage BAC-based physical map described in this paper are available to the research community.
Date: March 18, 2005
Creator: Gonzalez, Josefa; Nefedov, Michael; Bosdet, Ian; Casals, Ferran; Calvete, Oriol; Delprat, Alejandra et al.
Partner: UNT Libraries Government Documents Department

The BDGP gene disruption project: Single transposon insertions associated with 40 percent of Drosophila genes

Description: The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in more than 30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6,300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7,140 lines. It now includes individual lines predicted to disrupt 5,362 of the 13,666 currently annotated Drosophila genes (39 percent). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene mis-expression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.
Date: January 13, 2004
Creator: Bellen, Hugo J.; Levis, Robert W.; Liao, Guochun; He, Yuchun; Carlson, Joseph W.; Tsang, Garson et al.
Partner: UNT Libraries Government Documents Department

Large-scale turnover of functional transcription factor bindingsites in Drosophila

Description: The gain and loss of functional transcription-factor bindingsites has been proposed as a major source of evolutionary change incis-regulatory DNA and gene expression. We have developed an evolutionarymodel to study binding site turnover that uses multiple sequencealignments to assess the evolutionary constraint on individual bindingsites, and to map gain and loss events along a phylogenetic tree. Weapply this model to study the evolutionary dynamics of binding sites ofthe Drosophila melanogaster transcription factor Zeste, using genome-widein vivo (ChIP-chip) binding data to identify functional Zeste bindingsites, and the genome sequences of D. melanogaster, D. simulans, D.erecta and D. yakuba to study their evolution. We estimate that more than5 percent of functional Zeste binding sites in D. melanogaster weregained along the D. melanogaster lineage or lost along one of the otherlineages. We find that Zeste bound regions have a reduced rate of bindingsite loss and an increased rate of binding site gain relative to flankingsequences. Finally, we show that binding site gains and losses areasymmetrically distributed with respect to D. melanogaster, consistentwith lineage-specific acquisition and loss of Zeste-responsive regulatoryelements.
Date: July 14, 2006
Creator: Moses, Alan M.; Pollard, Daniel A.; Nix, David A.; Iyer, VenkyN.; Li, Xiao-Yong; Biggin, Mark D. et al.
Partner: UNT Libraries Government Documents Department

The histone H3K9 methylation and RNAi pathways regulate normalnucleolar and repeated DNA organization by inhibiting formation ofextrachromosomal DNAs

Description: In order to identify regulators of nuclear organization, Drosophila mutants in the Su(var)3-9 histone H3K9 methyltransferase, RNAi pathway components, and other regulators of heterochromatin-mediated gene silencing were examined for altered nucleoli and positioning of repeated DNAs. Animals lacking components of the H3K9 methylation and RNAi pathways contained disorganized nucleoli, ribosomal DNA (rDNA) and satellite DNAs. The levels of H3K9 dimethylation (H3K9me2) in chromatin associated with repeated DNAs decreased dramatically in Su(var)3-9 and dcr-2 (dicer-2) mutant tissues compared to wild type. We also observed a substantial increase in extrachromosomal repeated DNAs in mutant tissues. The disorganized nucleolus phenotype depends on the presence of Ligase 4 (Lig4), and ecc DNA formation is not induced by removal of cohesin. We conclude that H3K9 methylation of rDNA and satellites, maintained by Su(var)3-9, HP1, and the RNAi pathway, is necessary for the structural stability of repeated DNAs, which is mediated through suppression of non-homologous end joining (NHEJ). These results suggest a mechanism for how local chromatin structure can regulate genome stability, and the organization of chromosomal elements and nuclear organelles.
Date: June 15, 2006
Creator: Peng, Jamy C. & Karpen, Gary H.
Partner: UNT Libraries Government Documents Department

PointCloudExplore 2: Visual exploration of 3D gene expression

Description: To better understand how developmental regulatory networks are defined inthe genome sequence, the Berkeley Drosophila Transcription Network Project (BDNTP)has developed a suite of methods to describe 3D gene expression data, i.e.,the output of the network at cellular resolution for multiple time points. To allow researchersto explore these novel data sets we have developed PointCloudXplore (PCX).In PCX we have linked physical and information visualization views via the concept ofbrushing (cell selection). For each view dedicated operations for performing selectionof cells are available. In PCX, all cell selections are stored in a central managementsystem. Cells selected in one view can in this way be highlighted in any view allowingfurther cell subset properties to be determined. Complex cell queries can be definedby combining different cell selections using logical operations such as AND, OR, andNOT. Here we are going to provide an overview of PointCloudXplore 2 (PCX2), thelatest publicly available version of PCX. PCX2 has shown to be an effective tool forvisual exploration of 3D gene expression data. We discuss (i) all views available inPCX2, (ii) different strategies to perform cell selection, (iii) the basic architecture ofPCX2., and (iv) illustrate the usefulness of PCX2 using selected examples.
Date: March 31, 2008
Creator: International Research Training Group Visualization of Large and Unstructured Data Sets, University of Kaiserslautern, Germany; Institute for Data Analysis and Visualization, University of California, Davis, CA; Computational Research Division, Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA; Genomics Division, LBNL; Computer Science Department, University of California, Irvine, CA; Computer Science Division,University of California, Berkeley, CA et al.
Partner: UNT Libraries Government Documents Department

Widespread Discordance of Gene Trees with Species Tree inDrosophila: Evidence for Incomplete Lineage Sorting

Description: The phylogenetic relationship of the now fully sequencedspecies Drosophila erecta and D. yakuba with respect to the D.melanogaster species complex has been a subject of controversy. All threepossible groupings of the species have been reported in the past, thoughrecent multi-gene studies suggest that D. erecta and D. yakuba are sisterspecies. Using the whole genomes of each of these species as well as thefour other fully sequenced species in the subgenus Sophophora, we set outto investigate the placement of D. erecta and D. yakuba in the D.melanogaster species group and to understand the cause of the pastincongruence. Though we find that the phylogeny grouping D. erecta and D.yakuba together is the best supported, we also find widespreadincongruence in nucleotide and amino acid substitutions, insertions anddeletions, and gene trees. The time inferred to span the two keyspeciation events is short enough that under the coalescent model, theincongruence could be the result of incomplete lineage sorting.Consistent with the lineage-sorting hypothesis, substitutions supportingthe same tree were spatially clustered. Support for the different treeswas found to be linked to recombination such that adjacent genes supportthe same tree most often in regions of low recombination andsubstitutions supporting the same tree are most enriched roughly on thesame scale as linkage disequilibrium, also consistent with lineagesorting. The incongruence was found to be statistically significant androbust to model and species choice. No systematic biases were found. Weconclude that phylogenetic incongruence in the D. melanogaster speciescomplex is the result, at least in part, of incomplete lineage sorting.Incomplete lineage sorting will likely cause phylogenetic incongruence inmany comparative genomics datasets. Methods to infer the correct speciestree, the history of every base in the genome, and comparative methodsthat control for and/or utilize this information will be valuableadvancements for the field of comparative genomics.
Date: August 28, 2006
Creator: Pollard, Daniel A.; Iyer, Venky N.; Moses, Alan M. & Eisen,Michael B.
Partner: UNT Libraries Government Documents Department

Conservation patterns in different functional sequence categories of divergent Drosophila species

Description: We have explored the distributions of fully conservedungapped blocks in genome-wide pairwise alignments of recently completedspecies of Drosophila: D.yakuba, D.ananassae, D.pseudoobscura, D.virilisand D.mojavensis. Based on these distributions we have found that nearlyevery functional sequence category possesses its own distinctiveconservation pattern, sometimes independent of the overall sequenceconservation level. In the coding and regulatory regions, the ungappedblocks were longer than in introns, UTRs and non-functional sequences. Atthe same time, the blocks in the coding regions carried 3N+2 signaturecharacteristic to synonymic substitutions in the 3rd codon positions.Larger block sizes in transcription regulatory regions can be explainedby the presence of conserved arrays of binding sites for transcriptionfactors. We also have shown that the longest ungapped blocks, or'ultraconserved' sequences, are associated with specific gene groups,including those encoding ion channels and components of the cytoskeleton.We discussed how restrained conservation patterns may help in mappingfunctional sequence categories and improving genomeannotation.
Date: October 1, 2005
Creator: Papatsenko, Dmitri; Kislyuk, Andrey; Levine, Michael & Dubchak, Inna
Partner: UNT Libraries Government Documents Department

Resources for Biological Annotation of the Drosophila Genome

Description: This project supported seed money for the development of cDNA and genetic resources to support studies of the Drosophila melanogaster genome. Key publications supported by this work that provide additional detail: (1) ''The Drosophila gene collection: identification of putative full-length cDNAs for 70% of D. melanogaster genes''; and (2) ''The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes''.
Date: August 8, 2005
Creator: Rubin, Gerald M.
Partner: UNT Libraries Government Documents Department