Genome Biology and Biotechnology

Transcription

1 Genome Biology and Biotechnology Functional Genomics Prof. M. Zabeau Department of Plant Systems Biology Flanders Interuniversity Institute for Biotechnology (VIB) University of Gent International course 2005 Functional Genomics the Paradigm Shift Large-scale genome sequencing generates parts lists complete inventories of genes and functional elements A new challenge to understand the function of the many genes predicted In general 90% to 95% of the genes have unknown functions Genome sequencing has triggered a transition from vertical (reductionist) approaches to horizontal (large scale) approaches Each approach has its own strengths and weaknesses Reprinted from: Vidal M., Cell, 104, 333 (2001) Vertical Versus Horizontal Approach The vertical approach The vertical or reductionist approach Studies one or a few proteins or genes at a time by applying different experimental tools to test hypotheses Well proven by decades of research The reductionist approach is based on the principle of understanding the whole by studying selected parts The reductionist approach has severe limitations lacks efficiency In well-studied model organisms decades of hypothesis driven research has discovered only 5 to 10% of the genes Fails to give a comprehensive picture of biology The study of Gal4p provided a useful model of how transcription factors work but gives no insight in global transcriptional responses Reprinted from: Vidal M., Cell, 104, 333 (2001) The horizontal Approach The horizontal or large scale approach studies large numbers of genes or proteins in parallel using high-throughput tools Microarrays, systematic gene knock outs Instrumentation for automated and high-throughput analysis Robots: automated liquid handlers Automated data acquisition instruments: e.g. sequencers Well suited for massively parallel studies Large scale approach is limited lack of giving conclusive evidence Noisy data with high rates of false positives or negatives Observations must to be confirmed Functional gene maps Functional genomics can be regarded as functional mapping within two-dimensional matrices One axis corresponds to all genes of an organism The other axis represents a set of conditions to which the organism is exposed Experimental conditions, various mutant backgrounds Each omics approach represents a different map Conditions Omics Genes n

2 Functional Maps or -omes ORFeome Phenome Transcriptome Localizome DNA Interactome Interactome Conditions Proteome proteins Genes or proteins n Genes Mutational phenotypes Expression profiles Cellular, tissue location Protein-DNA interactions Protein interactions The basic rationale of functional genomics Functionally related genes share common properties Are likely to be coregulated at the transcriptional level Transcriptome maps consist of ''expression clusters'' of coregulated genes Loss-of-function mutations should confer similar or opposite phenotypes Phenome maps consist of sets of genes giving similar phenotypes or ''pheno-clusters'' Their protein products are likely to interact physically Interactome mapsconsist of networks of interacting proteins ''interaction clusters'' Their protein products are likely to localize in similar cellular compartments Have similar location in the localisome After: Vidal M., Cell, 104, 333 (2001) Integration of Functional Maps Functional maps provide a rough indication of gene function Integration of functional maps in a biological atlas overcomes this limitation by overlaying sets of functional characteristics Functional Genomics Functional Genomics provides the tools for Identifying the function of all genes overlaying sets of functional characteristics Functional maps provide lists of clusters that contain both characterized and uncharacterized genes Provides hypotheses for the function of uncharacterized genes Functional Genomics provides approaches for the ultimate understanding of life at the molecular level based on the description of Each protein individually and The interactions between the proteins involved in particular biological processes Reprinted from: Vidal M., Cell, 104, 333 (2001) Genome Biology and Biotechnology Functional Maps or -omes ORFeome Phenome Conditions Genes or proteins n Genes Mutational phenotypes 6. The ORFeome Transcriptome DNA Interactome Localizome Expression profiles Protein-DNA interactions Cellular, tissue location Interactome Protein interactions International course 2005 Proteome proteins After: Vidal M., Cell, 104, 333 (2001)

3 The ORFeome: Genes in the Genome The genome represents the basic compendium of all genes that make up an organism The ORFeome represents the basic compendium of all protein coding genes as defined by their Open Reading Frames (ORFs) Predicted ORFs must be validated In higher organisms gene identification is complicated by Intron / exon structure The ORFeome platforms provide Large scale approach for validating predicted genes high throughput recombinational cloning technology Resources for functional genomics projects Recombinational Cloning One step cloning technology Site specific recombination instead of restriction/ligation Not dependent on availability of restriction sites 100% efficient: only one recombinant DNA product without byproducts No cloning step needed: no need to assay independent clones Fully automatable simple pipetting in microtiter plates Very precise recombination system allowing high fidelity DNA engineering Versatile cloning technology Genes can be easily transferred into a range of vector systems Expression, Gene fusion, RNAi GATEWAY Recombinational Cloning Based on the bacterio phage lambda integration & excision system Phage lambda integration & excision system Integration Excision attl phage attb attp attb Bacterial genome attr Recombinational Cloning of ORFs Designer oligo start attb1 cdna attb1 Phage lambda integration: Integrase & bacterial IHF PCR ORF ORF TG Entry Vector stop attb2 attb2 TG - Toxic gene Recombinational Cloning of ORFs Recombinational Cloning of gene Fusions Destination vector Entry clone Destination vector attp1 attp2 attl attr attb1 attb2 DNA binding domain Activation domain Phage lambda integration: Integrase & bacterial IHF Phage lambda excision: Integrase, IHF & Exisionase attl1 attl2

4 GATEWAY Recombinational Cloning Second generation att sites and BP cloning First generation cloning technology DNA Cloning Using In Vitro Site-Specific Recombination Hartley et. al., Genome Research 10, (2000) Designed for large scale cloning of ORFs High throughput platform for generating ORFeome libraries Second generation technology Concerted Assembly and Cloning of Multiple DNA Segments Using In Vitro Site-Specific Recombination Cheo et. al., Genome Research 14: (2004) Designed for large scale production of multi-segment expression clones Synthetic attb and attp sites Int cut site Left arms Right arms 4 simultaneous reactions BP cloning Int cut site Reprinted from: Cheo et. al., Genome Research 14: (2004) Multi-segment recombination cloning Multi-Segment Expression Clones Two-segment cloning Three-segment cloning The expanded repertoire of recombination sites for Concerted cloning of multiple DNA segments in a predefined order, orientation, and reading frame Generates collections of functional elements in a combinatorial fashion Applications linkage of promoters to genes generation of fusion proteins assembly of multiple protein domains The technology has broad implications for gene function analysis. expression of multidomain proteins Reprinted from: Cheo et. al., Genome Research 14: (2004) Reprinted from: Cheo et. al., Genome Research 14: (2004) The ORFeome of C. elegans version 1.0 PCR amplification of C. elegans ORFs ORFeome cloning was first demonstrated in C. Elegans. Predicted ORFs are amplified by PCR from a highly representative cdna library using ORF-specific primers Cloned by GATEWAY recombination cloning The C. elegans genome sequence predicted 18,959 ORFs PCR products of identified genes PCR products of Untouched genes identified genes Untouched genes

5 Successful PCR for the ORFs analyzed Conclusions ORFeome strategy provides experimental evidence for structure of genes in C. elegans ORFeome resource for large scale functional genomics version v1.1 Attempted PCR amplification of the 19,477 ORFs cloned 10,623 (55%) in-frame ORFs ORF Sequence Tags improved C. elegans gene annotations corrected the internal gene structure of 20% of the ORFs. C. elegans ORFeome Version 3.1 The C. elegans ORFeome is an evolving resource Gene prediction improvements Classification of the 4232 repredicted and new ORFs Reprinted from: Lamesch et. al., Genome Research 14: (2004) Reprinted from: Lamesch et. al., Genome Research 14: (2004) Conclusions Cloning of a complete ORFeome is an iterative process requires multiple rounds of experimental validation together with gradually improving gene predictions (bioinformatics) the ORFeome resource provides further verification of the predicted gene structures Note that the procedure will not reveal alternatively spliced transcripts unless GATEWAY clones are cloned individually ORFeome projects now underway Human Arabidopsis Drosophila Versatile Gene-Specific Sequence Tags for Arabidopsis Functional Genomics Hilson et. al., Genome Research 14: (2004) Paper presents The creation of a collection of gene-specific sequence tags (GSTs) representing 21,500 Arabidopsis genes Gene-specific sequence tags (GST) Correspond to short (150bp to 500bp) segments of ORFs selected to have no significant similarity with any other region in the genome Synthesized by PCR amplification from genomic DNA The GSTs provide a resource for large-scale gene function studies in multicellular eukaryotes RNA interference Microarray transcript profiling Reprinted from: Lamesch et. al., Genome Research 14: (2004)

6 Graphical representation of GSTs GST production GST Predicted gene High throughput PCR High throughput verification Reprinted from: Hilson et. al., Genome Research 14: (2004) Reprinted from: Hilson et. al., Genome Research 14: (2004) GST cloning in GATEWAY vectors The Caenorhabditis elegans Promoterome Dupuy et. al., Genome Research 14: (2004) Paper presents The development of a genome-wide resource of C. elegans promoters characterize the expression patterns of all predicted genes expressing localization markers such as the green fluorescent protein (GFP). "localizome" maps should provide information on where (in what cells or tissues) genes are expressed when (at what stage of development or under what conditions) genes are expressed in what cellular compartments the corresponding proteins are localized Reprinted from: Hilson et. al., Genome Research 14: (2004) The C. elegans promoterome "promoters" correspond to upstream intergenic regions (IGR) region from the ATG of the ORF to the end of the preceding ORF PCR fragment upper size limit of 2 kb to ensure high cloning efficiency Overview of promoterome cloning procedure analysis of PCR products large-scale cloning of the promoterome ORF Reprinted from: Dupuy et. al., Genome Research 14: (2004) Reprinted from: Dupuy et. al., Genome Research 14: (2004)

7 Conclusion Applications of recombinational cloning Promoterome version 1.1 Resource of 6000 C. elegans promoters cloned in the MultiSite Gateway system Promoterome constitutes an equally valuable resource as the ORFeome Promoters can be easily transferred into Gateway Destination vectors to drive expression of markers such as GFP (promoter::gfp constructs) GFP fusion with ORFs available in ORFeome resources (promoter::orf::gfp constructs) Reprinted from: Dupuy et. al., Genome Research 14: (2004) Reprinted from: Dupuy et. al., Genome Research 14: (2004) Recommended reading Functional genomics The concept of a biological atlas Vidal M., Cell, 104, 333 (2001) ORFeome resource and analysis The ORFeome of C. elegans Reboul et. al., Nat. Genet. 27, 332 (2001) The ORFeome of Arabidopsis Hilson et. al., Genome Research 14: (2004) Further reading ORFeome analysis GATEWAY Recombinational Cloning Hartley et. al., Genome Research 10, (2000) Cheo et. al., Genome Research 14: (2004) Walhout et al, Science 287: 116 (2000) C. elegans ORFeome Lamesch et. al., Genome Research 14: (2004) C. elegans Promoterome Dupuy et. al., Genome Research 14: (2004)