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BYU BIO 465 - splicing-microarrays

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Alternative SplicingPowerPoint PresentationSlide 3Slide 4Slide 5Human GenomeImplicationsApplicationSlide 9SpliceosomeSlide 11Slide 12Splicing errorsWhy splicingSlide 15Microarrays For Alt. SplicingAffymetrix Microarrays For Alt. SplicingIdeal Microarray ReadingsMotivationSome Previous WorkMethodMicroarray DesignTechnical workPredicting Alternative SplicingSlide 25Gene Expression MeasurementGene Expression MicroarraysAffymetrix MicroarraysMicroarray Potential ApplicationsMicroarray Data Analysis TypesMicroarray Data Mining ChallengesAlternative SplicingAs an introduction to microarraysQuickTime™ and aTIFF (LZW) decompressorare needed to see this picture.QuickTime™ and aTIFF (LZW) decompressorare needed to see this picture.QuickTime™ and aTIFF (LZW) decompressorare needed to see this picture.QuickTime™ and aTIFF (LZW) decompressorare needed to see this picture.Human Genome•90,000 Human proteins, initially assumed near that number of genes (initial estimates 153,000)•The 1000 cell roundworm Caenorhabditis elegans has 19,500 genes, corn has 40,000 genes•Current estimates are 25,000 or fewer genes•Alternative splicing allows different tissue types to perform different function with same gene assortmentImplications•75% of human genes are subject to alternative editing•faulty gene splicing leads to cancer and congenital diseases.•gene therapy can use splicingApplication•We talked before about apoptotis when the cell determines it cant be repaired•Bcl-x is a regulator of apoptotis, is alternatively spliced to produce either Bcl-x(L) that suppresses apoptosis, or Bcl-x(S) that promotes it.QuickTime™ and aTIFF (LZW) decompressorare needed to see this picture.Spliceosome•Five snRNA molecules U1, U2, U3, U4, U5, U6 combine with as many as 150 proteins to form the spliceosome•It recognizes sites where introns begin and end –Cuts introns out of pre-mRNA –joins exonsQuickTime™ and aTIFF (LZW) decompressorare needed to see this picture.Spliceosome•The 5’ splice site is at the beginning of the intron, the 3’ site is at the end•The average human protein coding gene is 28000 nucleotides long with 8.8 exons separated by 7.8 introns•exons are 120 nucleotides long while introns are 100-100,000 nucleotides longSplicing errors•familial dysautonomia results from a single-nucleotide mutation that causes a gene to be alternatively spliced in nervous system tissue•The decrease in the IKBKAP protein leads to abnormal nervous system development (half die before 30)•> 15% of gene mutations that cause genetic diseases and cancers are caused by splicing errors.Why splicing•Each gene generates 3 alternatively spliced mRNAs•Why so much intron (1-2% of genome is exons)?•Mouse and human differences are almost all splicing•Half of the human genome is made up of transposable elements, Alus being the most abundant (1.4 million copies)–They continue to multiply and insert themselves into the genome at the rate of one insertion per 100 human births•mutations in the Alu can create a 5’ or 3’ site in an intron causing it to be an exon•This mutation doesn’t impact existing exons•It only has effect when it is alternatively spliced inQuickTime™ and aTIFF (LZW) decompressorare needed to see this picture.Microarrays For Alt. Splicing•Use short oligonucleotides•Get a guess at the rate of expression of the oligoExon 1Exon 3Exon 2 Exon 4 Exon 5AffymetrixMicroarrays For Alt. SplicingExon 1Exon 3Exon 2 Exon 4 Exon 5Exon 1 Exon 2 Exon 4 Exon 5Exon 1 Exon 3 Exon 5Isoform 1:Isoform 2:Probe typesConstitutiveJunction ExonUnique (“Cassette”)Ideal Microarray ReadingsExon 1 Exon 2 Exon 4 Exon 5Exon 1 Exon 3 Exon 5Isoform 1:Isoform 2:Probe typesConstitutiveExonJunctionUnique (“Cassette”)aabcdeProbeExpressiona b c d eMotivation•Why alternatively splice?•How does it affect the resulting proteins?•Look at domains:–High level summary of protein–~80% of eukaryotic proteins are multi-domain–Domains are big relative to an exonSome Previous Work•Signatures of domain shuffling in the human genome. Kaessmann, 2002.Intron phase symmetry around domain boundaries•The Effects of Alternative Splicing On Transmembrane Proteins in the Mouse Genome. Cline, 2004.Half of TM proteins studied affected by alt-splicing.Method•Predict Alternative Splicing•Predict Protein Domains•Look for effects of Alt-Splicing on predicted domains–“Swapping”–“Knockout”–“Clipping”Microarray Design•Genes based on mRNA and EST data in mouse•Mapped to Feb. 2002 mouse genome freeze•~500,000 probes (~66,000 sets)•~100,000 transcripts•~13,000 gene modelsTechnical workGenome SpacetranscriptsprobesProvided dataOverlapOverlapOverlapgene modelsE@NM_021320 cc-chr10-000017.82.0G6836022@J911445 cc-chr10-000017.91.1G6807921@J911524_RC cc-chr10-000018.4.0Probe to transcript mappingGenerated DataPredicting Alternative Splicing•Using mouse alt-splicing microarrays•Data from Manny Ares–8 tissues–3 replicates of each tissuePredicting Alternative Splicing•General Approach: Clustering, then Anti-ClusteringQuickTime™ and aTIFF (LZW) decompressorare needed to see this picture.107 ClustersDetail ViewGene Expression Measurement•mRNA expression represents dynamic aspects of cell •mRNA expression can be measured with latest technology•mRNA is isolated and labeled with fluorescent protein•mRNA is hybridized to the target; level of hybridization corresponds to light emission which is measured with a laserGene Expression MicroarraysThe main types of gene expression microarrays:•Short oligonucleotide arrays (Affymetrix);•cDNA or spotted arrays (Brown/Botstein).•Long oligonucleotide arrays (Agilent Inkjet);•Fiber-optic arrays•...Affymetrix Microarrays50um1.28cm~107 oligonucleotides, half Perfectly Match mRNA (PM), half have one Mismatch (MM)Raw gene expression is intensity difference: PM - MMRaw imageMicroarray Potential Applications•Biological discovery–new and better molecular diagnostics–new molecular targets for therapy–finding and refining biological pathways•Recent examples–molecular diagnosis of leukemia, breast cancer, ...–appropriate treatment for genetic signature–potential new drug targetsMicroarray Data Analysis Types•Gene Selection–find genes for therapeutic targets–avoid false positives (FDA approval ?)•Classification (Supervised)–identify disease –predict outcome / select best


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BYU BIO 465 - splicing-microarrays

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