March 5-9, 2003Chicago, Illinois

44th Annual Drosophila Research Conference

Community ResourcesI. Flybase Demonstrations

i) The Apollo Annotation Viewer and Editor

• Annotations are Release 3.1• 85% of the gene annotations changed since Release 1• Download the Apollo Userguide; last updated Feb.27, 2003; contains info for developers• can save Apollo download as a GAME.XML file which can be edited and saved locally• Blue = “annotation” zone; white (or black) = “evidence” zone• Editing: can add comments, edit 5’ and 3’ ends, move “evidence” to “annotations”• Affymetrix oligo locations are available

Q: can you add your own data to the “evidence” zone?A: Not yet but it is a current priority (within the year)

Q: Is pseudoobscura data available here?A: Not yet, but will be integrated.

Flybase Demo continuedii) Bulk Data Retrieval and Advanced Queries

Flybase Batch Queries:

• can retrieve data in a number of formats, eg. HTML, tab delimited•Can obtain batch reports by ID

•e.g. submit a list of Ids( eg. CG #s) and get back a report containing selected fields from Flybase

• Annotation downloads (from Gadfly)•E.g. can retrieve gene sequences + 500bp upstream and downstream sequence

Q: Is it possible to download the sequence coordinates for all the ESTs?A: Intend to add downloadable GAME.XML files that will have all the “evidence” including EST coordinates. Currently not available BUT if we email to Queryhelp, they will help us obtain this info.

Techniques WorkshopI. Pseudoobscura sequenceSteven Richards, Baylor College of Medicine, Houston, TX

Genomic Sequencing started Nov. 2002Ave contig size: 18.7 kbNumber of contigs: 6,800 (6,000 in scaffolds)Still do do: sequence BAC ends; also an embryonic cDNA library in progressCan download all data from FTP site

http://pipeline.lbl.gov/pseudo Inna DubchakVista alignment between melanogaster and pseudoobscura sequenceCan view/download sequences and alignments

Did tBLASTn (protein query vs translated db) of 13,380 melanogaster proteins vs. obscura database: Found putative orthologs for 95% of the proteins

II. Patterns of Gene Expression in Drosophila (Flybase/BDGP)

• in situ and Affy expression data (in graph format) available

• Mar 2003 release: 2999 genes

• 1712 documented by 33,699 photos

• Now orienting all newly added photos along A/P axis

• Starting to analyze gene expression patterns in

pseudoobscura embryos

• Probe and Affy oligo locations are now available on a

gene map

III. “Flytrap”flytrap.med.yale.du

Collaboration between Cooley, Chia and Spradling labsProtein trapping using GFP:•mobilize a transposing containing a GFP “exon”; it has no initiator methionine so cannot be translated unless inserted into a gene• contains splice sequences so will be included if inserted into an intronProblems: •P insertions into coding regions are infrequent•insertion has to be in correct reading frame with respect to the inserted proteinTherefore, use automated embryo sorter (Union Biometra) + FACs analyses

•Screened 3 million embryos•Recovered 2,000 GFP+ embryos• 70% survive; half of these carry transposase and are discarded•600 lines in database (most/all have ovary expression but other tissues not investigated yet)

• Useful for gene discovery (*e.g. inxs/echinus candidate genes) and generation of mutations

Basic Expression Search Tool (BEST)

Dudhir Kumar, Arizona State University

•A “BLAST”-like program for expression patterns

•Finds matches based on spatial expression (can select “identical” or

“overlapping”)

•Data from literature and BDGP expression project

•Size of embryo is standardized and pattern is extracted as a binary image

•Greyscale work will also be included for BDGP images

“Fly Express” Database at FlyExpress.net (not yet available!)

Second Generation Deficiency Kit

• DrosDel Consortium of European labs; established 1999

• Use RS P elements (Golic) to create FRT-mediated deficiencies•Distributed by Szeged Stock Centre• http://www.drosdel.org.uk

Gene Disruption Project (BDGP)

Allan Spradling, Carnegie Institution

•Phase 2 (Baylor, Berkeley, Carnegie, Bloomington, LBNL)• now 6400 lines•Re-analysing P elements using Release 3.1 (in progress)•Renewed funding so expect >10,000 new lines in the next 4 years

Techniques/Genomics SessionI.Characterizing the heritability of gene expression using whole transcriptome Agilent long oligonucleotide microarrays (G. Gibson, North Carolina State U)

Whole transcriptome arrays consisting of 60-mer target long oligonucleotidessynthesized in situ using inkjet SurePrint technologies

22,000 “features” using Version 3 annotation; one third of the genes represented by two different oligos

II. “Flymove” (http://flymove.uni-muenster.de)All available for download:• Images of embryonic images• In vivo movies of embryo development• Histological sections• Organogenesis (salivary glands!)• Genetics• Life Cycle

III. Functional anlaysis of gene and protein networks in Drosophila based on microarray and genome wide RNAi studies

M. Hild, Heidelberg, Germany (Paro lab)

•Generated a new annotation (“Heidelberg annotation”) based on predictionsmade by the BDGP and Sanger Centre (Victor Solovej ?)•Identified 20,396 putative ORFs•Designed primers for microarrays(two copies of the 20,903 PCR products, 70 long oligos for smaller ORFs and 64 different controls •Primers available commercially: “Eurogentec array” (95% genes represented)•Did profiling from 9 different stages of Drosophila life cycle:

Array data indicates presence of additional 3,500 genes (compared to BDGP)So new estimate is 16,500-17,500 genesConfirming array data by in situ hybridization for all the “new” genes

Also used PCR products for an RNAi screen (added T7 to “2nd” set of primers;384-well format) for cell death and growth defects:Found 450 dsRNAs with a phenotype in a cell death/survival assay(104 of these dsRNAs are from Sanger annotations)

IV. Cell based Genome-wide RNAi screens in Drosophila

M. Boutros, Harvard (Perrimon Lab)

•Started 2 years ago•Synthesized 21,300 dsRNAs for high-throughput RNAi screens(gene specific primers and then ten different primers to amplify all sequences; add T7 to primer ends)•Based on Heidelberg annotation•Add dsRNA to 384-well plates; seed cells on top; conduct assay•Assays involve read-outs that can be monitored by luminescent or fluorescent read outs using a plate reader (e.g. reporter genes), or visualphenotypes monitored by automatic microscopy

e.g. cell death screen: control – thread (iap1) RNAi (loss of thread leads to increased cell death)•Found about 50 dsRNAs with a thread-like phenotype (phenocopied th)•60% of identified modifiers have no GO annotation•Annotated genes include ubiquitin (Ubi-p63E) and ubiquitin-like genes (CG11700)

Salivary gland Induction of Glue genes in mid-third instar requires a functional ecdysonereceptor ( Andy Andres and John Merriam)

•Glue proteins are synthesized early in larval development and stored inSecretory granules

•Glue proteins secreted into duct in response to ecdysone signaling•Animals then expel the glue (and immobilize themselves as pupae)

•Made sgs-GFP construct = Glue-GFP (cool pictures)

•Made Glue-Gal4; UAS-EcR-DN and this blocked secretion•Used hs-Gal4 ( this is “leaky” in sgs and recommend this as the “best” salivary gland specific driver (fkh-Gal4 also good)

•Used hs-Gal4; UAS-EcR-DN to block EcR signaling and thus cell death;•Could see GFP labeled sgs stuffed up into thorax or head; adults appearedto be okay

Q: Is there a function known between the prepupal glue secretion and histolysis?

A: Late genes (L71) maybe produce antimicrobial genes and these become distributed through the animal to protect during histolysis (timing would be consistent with gut histolysis only)

Also hypothesized that many other proteins secreted into the lumen throughout but no really good evidence (?) (see old Hogness papers)

Eric – colleague who thinks that antimicrobial peptides have a role in alteringmembranes, e.g. during bacterial infections-could be doing the same thing is SGs?

-Immunity person: perhaps AMPs have a role in sclerotization (I.e. hardening/ tanning of the cuticle)

Salivary glands and AntiMicrobial Proteins

DHR4(Kirst King-Jones, Carl Thummel Lab)

Drosophila Hormone Receptor 4Orphan nuclear receptor; maps to 2C

Expressed in embryo, 2nd instar and 0-6 hr prepupa

P element allele: hypomorph (mRNA greatly reduced); P insertion is 2 kb from the 5’ end.

Lethal phase analysis: die as prepupae; pupal cases shaped abnormally,Not all evert spiracles, decreased tanning and growth

Saw appearance of “dwarf” larvae; analyzed mouth hooks to identify them as3rd instars

Made a UAS-DHR4 RNAi line to further analyze phenotypes; see failedeclosion attempts (muscle defect?)

Autophagy in Drosophila( Poster 265A, R. Scott and T.P. Neufeld, University of Minnesota)

Aut7-GFPApg5-GFPLocalize to punctate structures, thought to be autophagosomes

P element disruptions:Analyze phenotype in fat body by inducing aa starvation (feed sugar water)and visualizing acidic vesicles with Lysotracker staining (Molecular Probes); Stains autophagosomes (Munafo and Colombo, 2001)Apg4 (viable) – no phenotype (I.e. like wt; high levels of lysotracker)Apg1 (pupal lethal) – decreased LysotrackerAut10 (2nd instar lethal) – decreased Lysotracker

(did not have the P element alleles listed but obtained from P projects)

After starvation, there is an initial “burst” of Lysotracker staining and then itdecreases to a “steady” level (above feeding levels which were not detectable)Q: Can you induce/maintain a high level? And would this result in autophagic cell death??

Functional roles for reaper, hid and diap1 during Drosophila metamorphosis

Poster 1065C, V. Yin, C. Thummel

PKA activity is required for postmetamorphic programmedcell death in wing epidermal cells

Poster 950B, (Kimura, and Oota, Japan)

Genetic and molecular characterization of the morgue genePoster 953B, Schraeder, Wang and Nambu, U of Massachusetts

Cell lines in Drosophila

Cell line Source Suspension or attached

Trans-fectable

RNAi Does Rpr induce blebbing

Phagocytic

S2 Embryonic hemocytes

Lightly attach

Y Y Y Y; efficient

SL2 Embryonic hemocytes

Lightly attach

Y Y Y; efficient

MBN2 Malignant larval hemocytes

Lightly attach

Y Y Y

DL2 Lightly attach

Y Y N or weak

Kc Embryonic

Hemocytes

Lightly attach

Y Y Y; larger blebs?

Y

Clone 8 disc Attach strongly

Y Y? ?

Poster 964A Phagocytosis; K. White lab