Bài giảng Molecular Biology - Chapter 25 Genomics II: Functional Genomics, Proteomics, and Bioinformatics

Molecular BiologyFifth EditionChapter 25Genomics II: Functional Genomics, Proteomics, and BioinformaticsLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.125.1 Functional Genomics: Gene Expression on a Genomic ScaleFunctional genomics refers to those areas that deal with the function or expression of genomesAll transcripts an organism makes at any given time is an organism’s transcriptomeUse of genomic information to block expression systematically is called genomic functional profilingStudy of structures and functions of the protein products of genomes is proteomics 2TranscriptomicsThis area is the study of all transcripts an organism makes at any given timeCreate DNA microarrays and microchips that hold 1000s of cDNAs or oligosHybridize labeled RNAs from cells to these arrays or chipsIntensity of hybridization at each spot reveals the extent of expression of the corresponding geneMicroarray permits canvassing expression patterns of many genes at onceClustering of expression of genes in time and space suggest products of these genes collaborate in some process3Oligonucleotides on a Glass Substrate4Serial Analysis of Gene ExpressionSerial Analysis of Gene Expression (SAGE) allows us to determine:Which genes are expressed in a given tissueThe extent of that expressionShort tags, characteristic of particular genes, are generated from cDNAs and ligated together between linkersThese ligated tags are then sequenced to determine which genes are expressed and how abundantly5SAGE6Cap Analysis of Gene Expression (CAGE)CAGE gives the same information as SAGE about which genes are expressed and how abundantly, in a given tissueIt focuses on the 5’-ends of mRNAs, which allows for the identification of transcription start sites and may help in locating pormoters7Whole Chromosome Transcription MappingHigh density whole chromosome transcriptional mapping studies have shown a majority of sequences in cytoplasmic poly(A)RNAs derive from non-exon regions of human chromosomesAlmost half of the transcription from these same chromosomes is nonpolyadenylatedResults indicate that great majority of stable nuclear and cytoplasmic transcripts in these chromosomes come from regions outside exonsHelps to explain the great differences between species whose exons are almost identical8Transcription maps of 10 Human Chromosomes9Genomic Functional ProfilingGenomic functional profiling can be performed in several waysA type of mutation analysis, deletion analysis - mutants created by replacing genes one at a time with antibiotic resistance gene flanked by oligomers serving as barcode for that mutantA functional profile can be obtained by growing the whole group of mutants together under various conditions to see which mutants disappear most rapidly10RNAi AnalysisAnother means of genomic functional analysis on complex organisms can be done by inactivating genes via RNAiAn application of this approach targeting the genes involved in early embryogenesis in C. elegans has identified:661 important genes326 are involved in embryogenesis11Tissue-Specific Functional ProfilingTissue-specific expression profiling can be done by examining a spectrum of mRNAs whose levels are decreased by an exogenous miRNAThen compare to the spectrum of expression of genes at the mRNA level in various tissuesIf that miRNA causes a decrease in the levels of mRNAs naturally low in cells expressing the miRNASuggests that the miRNA is at least a partial cause of those natural low levelsThis type of analysis has implicated miR-124 in destabilizing mRNAs in brain tissuemiR-1 in destabilizing mRNAs in muscle tissue12Locating Target Sites for Transcription FactorsChIP-chip analysis can be used to identify DNA-binding sites for activators and other proteinsSmall genome organisms - all of the intergenic regions can be included in the microarrayIf genome is large, that is not practicalTo narrow areas of interest can use CpG islandsThese are associated with gene control regionsIf timing/conditions of activator’s activity are known, control regions of genes known to be activated at those times, or under those conditions, can be used13Locating Target Sites for Transcription FactorsTag sequencing, or ChIPSeq, in which chromatin pieces precipitated by ChIP are repeatedly sequenced, can also be used to identify transcription factor-binding sitesKnowledge of the sequence of multiple mammalian genomes allows one to narrow the search for human transcription factor binding sites by beginning with conserved regions of the genomeIn addition, it is easier to search for cis-regulatory modules (CRMs), which contain several transcription factor binding sites14Locating enhancers that bind unknown proteinsThere are still many enhancers whose protein partners are unknownPennachio and colleagues started the search for vertebrate enhancers by looking for highly conserved non-coding DNA regions in 2006The strategy had a remarkably high success rate but has a drawback in that it only detects highly conserved sequences and not all important control regions are conserved15Locating promoters that bind unknown proteinsRen and colleagues performed a genome-wide search for human promoters and were surprised to find that many genes have alternative promoters located hundreds of bases away from their primary promotersClass II promoters can be identified using ChIP-chip analysis with an anti-TAF1 antibodyIn one study using human fibroblasts, over 9,000 promoters were identified and over 1600 genes had multiple promoters16In Situ Expression AnalysisThe mouse can be used as a human surrogate in large-scale expression studies that would be ethically impossible to perform on humansScientists have studied the expression of almost all the mouse orthologs of the genes on human chromosome 21Expression followed through various stages of embryonic developmentCatalogued the embryonic tissues in which these genes are expressed17Single-Nucleotide Polymorphisms (SNPs)Single-nucleotide polymorphisms can probably account for many genetic conditions caused by single genes and even some by multiple genesMight be able to predict response to a drugHaplotype map with over 1 million SNPs makes it easier to sort out important SNPs from those with no effect18Structural VariationStructural variation is a prominent source of variation in human genomesInsertionsDeletionsInversionsRearrangements of DNA chunksSome structural variation can, in principle, predispose certain people to contract diseasesSome variation is presumably benignSome also is demonstrably beneficial1925.2 ProteomicsThe sum of all proteins produced by an organism is its proteomeStudy of these proteins, even smaller subsets, is called proteomicsSuch studies give a more accurate picture of gene expression than transcriptomics studies do20Protein Separations and AnalysisCurrent research in proteomics requires first that proteins be resolved, sometimes on a massive scaleBest tool for separation of many proteins at once is 2-D gel electrophoresisAfter separation, proteins must be identifiedBest method of identification involves digestion of proteins one by one with proteasesThen identify the peptides by mass spectrometryIn the future, microchips with antibodies attached may allow analysis of proteins in complex mixtures without separation21Quantitative ProteomicsTo determine the changes in protein levels upon perturbation of a cell culture, one can label the cells under the first condition with a light isotopic tag, and under the second condition with a heavy isotopic tagIf the proteins are labeled in vivo, the cell cultures can be mixed, the proteins can be extracted and fragmented by proteolysis and upon further separation can be subjected to mass spectronomyThe ratio of heavy to light peak areas will reflect the change in protein concentration as the growth conditions change22Comparative ProteomicsWhat makes a worm a worm and a fly a fly?Mass spectrometry data can be used to compare protein concentrations in two different organismsThis type of analysis was applied to C.elegans and Drosophila to reveal that the concentrations of orthologous proteins are correlated much better than the orthologous mRNAs in the two organisms23Protein InteractionsMost proteins work with other proteins to perform their functionsSeveral techniques are available to probe these interactionsYeast two-hybrid analysis has been used for some time, now other methods are availableProtein microarraysImmunoaffinity chromatography with mass spectrometryOther combinations24Detecting Protein-Protein Interactions2525.3 BioinformaticsBioinformatics involves the building and use of biological databasesSome of these databases contain the DNA sequences of genomesEssential for mining the massive amounts of biological data for meaningful knowledge about gene structure and expression26Finding Regulatory Motifs in Mammalian GenomesUsing computational biology techniques, Lander and Kellis have discovered highly conserved sequence motifs in 4 mammalian species, including humans:In the promoter regions, these motifs probably represent binding sites for transcription factors3’-UTRs motifs probably represent binding sites for miRNAs27Using the DatabasesThe National Center for Biological Information (NCBI) website contains a vast store of biological information, including genomic and proteomic dataStart with a sequence and discover the gene to which it belongs, then compare that sequence with that of similar genesQuery the database with a topic for information View structures of protein in 3D by rotating the structure on your computer screen28