Transcript lecture 4
Limitations of genome projects Windowjhgjhddoorhubbahubbastairduh 107 3.109 What do proteins do for a living? Post-genomics (A) Identifying genes from the sequence (B) Gene expression profiling (C) Genome activity studies Genomes2 by TA Brown; chapter 7 (A) Hunting genes from the sequence 2 broad approaches 1) Ab initio method (computational) 2) Experimental method Ab initio method (computational) Scanning ORFs (open reading frames) – initiation or termination codons Codon bias found in specific species Exon-intron boundaries Upstream control sequences – e.g conserved motifs in transcription factor binding regions CpG islands Homology searches Ab initio method (computational)….. Software for automated annotation of genes like GENSCAN, Genie, GENEBUILDER etc are being used. These scan for special features like 1) Scanning ORFs (open reading frames) – initiation or termination codons AAC TAA ATG 5’- ATGACGCATGATCGAGGAT –3’ 3’ – TACTGCGTACTAGCTCCTA –5’ CTA CCT TCC Ab initio method (computational)… Codon bias found in specific species Not all codons used at same frequency e.g.human leucine mainly coded by CTG and rarely by TTA or CTA Exon-intron boundaries (splice sites) 5’-AG GTAAGT-3’ hit and miss affair Upstream control sequences – e.g conserved motifs in transcription factor binding regions CpG islands experimental method Experimental evaluation based on the use of transcribed RNA to locate exons and entire genes from DNA fragment. experimental method 2 main strategies Hybridisation approaches – Northern Blots, cDNA capture / cDNA select, Zoo blots Transcript mapping: RT-PCR, exon trapping etc In this method, known DNA databases are searched to find out whether the test sequence is similar to any other known genes, suggesting an evolutionary relationship. Northern Blot Fig 7.4: Genomes 2 Zoo Blot Fig 7.5: Genomes 2 RT-PCR Fig 7.: Genomes 2 Exon trapping Fig 7.8: Genomes 2 (B) Gene expression profiling • COMPUTATIONAL APPROACH Homology searches for either - Orthologous genes (homologues in different organisms with common ancestor) - Paralogous genes (genes in the same organism, e.g. multigene families) (B) Gene expression profiling….. • EXPERIMENTAL APPROACH gene inactivation methods (knockouts, RNAi, site-directed mutagenesis, transposon tagging, genetic footprinting etc) Gene overexpression methods (knock-ins, transgenics, reporter genes etc) (C) Genome activity studies Gene expression needs to be complemented by Transcriptome analysis Proteome analysis The transcriptome Total RNA Non-coding RNA (96%) coding RNA (4%) Pre-r RNA Pre-t RNA hn RNA sn RNA sc RNA sno RNA mRNA r RNA t RNA All organisms eukaryotes bacteria tm RNA etc The transcriptome complete collection of transcribed elements of the genome transcriptome maps will provide clues on Regions of transcription • Transcription factor binding sites • Sites of chromatin modification • Sites of DNA methylation • Chromosomal origins of replication The transcriptome Analysis can be done by either SAGE (serial analysis of gene expression) technology Microarray technology SAGE Shortcut to doing cDNA library screening SAGE tags identify • mRNAs derived from known genes • anonymous mRNAs, also known as expressed sequence tags (ESTs) • mRNAs derived from currently unidentified genes Advantages • Analyzes all transcripts (Transcriptome) without prior selection of known genes • Provides quantitative data on both known and unknown genes • Ideally suited for determining changes on gene expression as consequence of an experimental treatment (e.g. carcinogen, hormone) SAGE Microarrays – allows comparisons Microarrays…. Proteomics Proteomics Nature (2003) March 13: Insight articles from pg 194 Proteomics Proteome projects - co-ordinated by the HUPO (Human Protein Organisation) Involve protein biochemistry on a highthroughput scale Problems limited and variable sample material, sample degradation, abundance, post-translational modifications, huge tissue, developmental and temporal specificity as well as disease and drug influences. Nature (2003) March 13: Insight articles from pgs 191-197. Approaches in proteomics High throughput approach 1) Mass- spectrometry based 2) Array based 3) Structural proteomics 4) Informatics 5) Clinical proteomics Nature (2003) March 13: Insight articles from pgs 191-197. High throughput approaches in proteomics 1) Mass spectrometry-based proteomics: relies on the discovery of protein ionisation techniques. used for protein identification and quantification, profiling, protein interactions and modifications. Nature (2003) March 13: Insight articles from pgs 191-197 Mass spectrometry (MS) Nature (2003) March 13: Insight articles from pgs 191-197 Principle of MS oion source, omass analyser that measures mass-to-charge ratio (m/z) odetector that registers the number of ions at each m/z value Electrospray ionisation (ESI) matrix-assisted laser desortion/ionisation (MALDI) MALDI-MS - simple peptide mixtures whereas ESI-MS - for complex samples. Nature (2003) March 13: Insight articles from pgs 191-197. Principle of MALDI-TOF Matrix assisted laser desorption/ ionisation – time of flight Fig 7.24 Genomes 2 by TA Brown pg 210 2) Array-based proteomics Based on the cloning and amplification of identified ORFs into homologous (ideally used for bacterial and yeast proteins) or sometimes heterologous systems (insect cells which result in post-translational modifications similar to mammalian cells). A fusion tag (short peptide or protein domain that is linked to each protein member e.g. GST) is incorporated into the plasmid construct. Nature (2003) March 13: Insight articles from pgs 191-197. Array based proteomics…. a. Protein expression and purification b. Protein activity: Analysis can be done using biochemical genomics or functional protein microarrays. c. Protein interaction analysis two-hybrid analysis (yeast 2-hybrid), FRET (Fluorescence resonance energy transfer), phage display etc d. Protein localisation: immunolocalisation of epitope-tagged products. E.g the use of GFP or luciferase tags Nature (2003) March 13: Insight articles from pgs 191-197. 3) Structural proteomics ! a. Protein expression and purification b. Protein activity: Analysis can be done using biochemical genomics or functional protein microarrays. c. Protein interaction analysis two-hybrid analysis (yeast 2-hybrid), FRET (Fluorescence resonance energy transfer), phage display etc d. Protein localisation: immunolocalisation of epitope-tagged products. E.g the use of GFP or luciferase tags Nature (2003) March 13: Insight articles from pgs 191-197. PROTEIN INTERACTION MAPS FOR MODEL ORGANISMS Nature Reviews Molecular Cell Biology 2; 55-63 (2001); doi:10.1038/35048107 Challenges for the future – ‘physiome’ Nature Reviews Molecular Cell Biology 4; 237-243 (2003)