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Ashwini Oke

Microarray data analysis towards the understanding the role of hzy in the formation of rhabdomeres. Ashwini Oke School of Informatics, Indiana University. Ashwini Oke. Drosophila compound eye. Montell, C. 1999. Rhabdomeres carry out the light reaction. ?. ?. Formation. Initiation.

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Ashwini Oke

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  1. Microarray data analysis towards the understanding the role of hzy in the formation of rhabdomeres Ashwini Oke School of Informatics, Indiana University Ashwini Oke

  2. Drosophila compound eye

  3. Montell, C. 1999 Rhabdomeres carry out the light reaction

  4. ? ? Formation Initiation Elongation Maintenance

  5. hzy is required for rhabdomere formation and phototransduction Development 130, 4383-4392 (2003)

  6. hzy is a transcription factor

  7. The phototransduction pathway The EMBO Journal (2001) 20, 4414–4422, doi:10.1093/emboj/20.16.4414

  8. Known targets of hzy

  9. Goals • Identify targets of hzy using oligonucleotide expression arrays • Determine the stage of rhabdomere formation that requires hzy • Identify DNA binding site for hzy • Understand the regulation of hzyitself

  10. Identifying regulatory targets of hzy

  11. Microarray design DGRC-2 Oligonucleotide Arrays Genotypes: WildType - cnbw Mutant - hzy - otd;hzy Time Points: 48 hr APF 72 hr APF 96 hr APF Replicates: 4 independent samples for each time point www.scq.ubc.ca/wp-content/cDNAarray.gif

  12. DGRC-2 arrays Probes ~14,400 probes representing 93.5% of the genome Negative Controls Blank Spotting_Solution Exogenous targets Positive Controls Housekeeping genes ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo ooooooooooooooooooo

  13. tiff image from the scanner

  14. GenePix Pro records spot intensities

  15. GenePix Pro creates .gpr file

  16. Analysis workflow R2.5.1 Bioconductor Packages: LIMMA, OLIN, MARRAY

  17. Preprocessing Scanned Image Heat Plot Density plot

  18. Normalization Genes Blank Spotting Solution Arabidopsis A = log 2 average intensity for the two channels M = log ratio of intensities for the two channels

  19. Normalization within array LOESS Local polynomial regression fit

  20. Normalization between arrays LIMMA: Linear models for Microarray Data Raw within array normalization between arrays normalization

  21. Density Plots after normalizations

  22. Differential analysis

  23. Volcano plot

  24. at 72 hr APF at 96 hr APF (12 total) (28 total)

  25. Gene Ontology GO Database provides a vocabulary to describe gene and gene products • Cellular Component Eg : Cytoplasmic, Nuclear, plasma membrane • Molecular Function Eg : Enzyme activity, protein binding • Biological Process Eg : Apoptosis, cell adhesion, phototransduction

  26. Classifying genes by GO At 72 hr APF At 96 hr APF

  27. otd : Another transcription factor regulating rhabdomere formation cnbw hzy otd cnbw hzy;otd

  28. Results from microarray analysis of hzy;otd double mutant

  29. hzy is required during elongation

  30. Identifying DNA binding site for hzy

  31. hzy has been shown to bind to a motif upstream of eyeG TAATNNNATTA Development 130, 4383-4392 (2003)

  32. MEME MEME is a tool for discovering motifs in a group of related DNA or protein sequences Primary targets of hzy should have a conserved binding site in their promoter region

  33. Motif search : 72 APF targets Down regulated genes meme 72.fas -dna -nmotifs 5 -mod zoops -revcomp -minw 3 -maxw 15

  34. A true motif should be conserved • For each of the hzy targets at 72 hr APF, find orthologs in other groups of Drosophila • Retrieve 500 b upstream of each gene • Run MEME on promoter regions from each specie

  35. Motif is conserved across orthologs

  36. Understanding the regulation of hzy

  37. Locating the promoter region 1.4 kb 0.8 kb Apitz, H. 2002. Dros.Inf.Serv.85:118-120.

  38. Promoter testing with Gal4-UAS system X Promoter Gal4 UAS Reporter

  39. Conserved regions

  40. Future Directions • RT PCR to confirm targets • Search hzy promoter region for known TFBS

  41. Thank you • Dr. Andrew Zelhof • Dr. Haixu Tang • Dr. Qunfeng Dong • MonalisaMishra PhD • Andreas Rechsteiner PhD • Jacqueline Lopez

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