Genome Biology and Biotechnology
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Transcript of Genome Biology and Biotechnology
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Genome Biology and Genome Biology and BiotechnologyBiotechnology
Functional GenomicsFunctional Genomics
International course 2005International course 2005
Prof. M. ZabeauProf. M. ZabeauDepartment of Plant Systems Biology Department of Plant Systems Biology
Flanders Interuniversity Institute for Biotechnology (VIB)Flanders Interuniversity Institute for Biotechnology (VIB)University of GentUniversity of Gent
International course 2005International course 2005
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Functional Genomics – the Paradigm Functional Genomics – the Paradigm ShiftShift
¤ 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)
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Vertical Versus Horizontal ApproachVertical Versus Horizontal Approach
Reprinted from: Vidal M., Cell, 104, 333 (2001)
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The vertical approachThe 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
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The horizontal ApproachThe 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
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Functional gene mapsFunctional 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
Genes
Omics
1 2 3 4 5 n
“Conditions”
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Functional Functional MapsMaps
or “-omes”or “-omes”
proteins
ORFeome
Localizome
Phenome
Transcriptome
Interactome
Proteome
Genes or proteins
Genes
Mutational phenotypes
Expression profiles
Protein interactions
1 2 3 4 5 n
DNA Interactome Protein-DNA interactions
“Conditions”
After: Vidal M., Cell, 104, 333 (2001)
Cellular, tissue location
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TheThe basic rationale of functional basic rationale of functional genomicsgenomics
¤ 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 maps consist of networks of interacting proteins
''interaction clusters''
– Their protein products are likely to localize in similar cellular compartments
• Have similar location in the localisome
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Integration of Functional MapsIntegration 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
Reprinted from: Vidal M., Cell, 104, 333 (2001)
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Functional GenomicsFunctional 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
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Genome Biology and Genome Biology and BiotechnologyBiotechnology
6. The ORFeome 6. The ORFeome
International course 2005International course 2005
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Functional Functional MapsMaps
or “-omes”or “-omes”
proteins
ORFeome
Localizome
Phenome
Transcriptome
Interactome
Proteome
Genes or proteins
Genes
Mutational phenotypes
Expression profiles
Protein interactions
1 2 3 4 5 n
DNA Interactome Protein-DNA interactions
“Conditions”
After: Vidal M., Cell, 104, 333 (2001)
Cellular, tissue location
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The ORFeome: Genes in the Genome 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
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Recombinational CloningRecombinational 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
Reprinted from: Walhout et al, Science 287: 116 (2000)
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Phage lambda integration & excision Phage lambda integration & excision systemsystem
attP
attB
attB
attL attR
phage
Bacterial genome
Excision
Integration
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Recombinational Cloning of ORFsRecombinational Cloning of ORFs
cDNA
start stop
Phage lambda integration:Integrase & bacterial IHF
attB2
attB1
ORF
Reprinted from: Walhout et al, Science 287: 116 (2000)
PCR
TG
TG - Toxic gene
Entry Vector
attB2attB1
Designer oligo ORF
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Recombinational Cloning of ORFsRecombinational Cloning of ORFs
Reprinted from: Walhout et al, Science 287: 116 (2000)
Phage lambda integration:Integrase & bacterial IHF
attB1
attP2
attL1 attL2
attP1
attB2
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Recombinational Cloning of gene FusionsRecombinational Cloning of gene Fusions
Phage lambda excision:Integrase, IHF & Exisionase
attL attR
DNA bindingdomain
Activationdomain
Reprinted from: Walhout et al, Science 287: 116 (2000)
Destination vector Entry clone Destination vector
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GATEWAY Recombinational CloningGATEWAY Recombinational Cloning
¤ First generation cloning technology – DNA Cloning Using In Vitro Site-Specific Recombination
• Hartley et. al., Genome Research 10, 1788-1795 (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 14:2111-2120 (2004)
– Designed for large scale production of multi-segment expression clones
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Second generationSecond generation att att sites and BP sites and BP cloningcloning
Int cut site
BP cloning
Int cut site
Left arms Right arms
Synthetic attB and attP sites
4 simultaneous reactions
Reprinted from: Cheo et. al., Genome Research 14:2111-2120 (2004)
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Multi-segment recombination Multi-segment recombination cloning cloning
Reprinted from: Cheo et. al., Genome Research 14:2111-2120 (2004)
Two-segment cloning Three-segment cloning
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Multi-Segment Expression Clones Multi-Segment Expression Clones
¤ 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 14:2111-2120 (2004)
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The ORFeome of C. elegans The ORFeome of C. elegans version version 1.01.0
¤ 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
Reprinted from: Reboul et. al., Nat.Genet. 27, 332 (2001)
9.503 identified genes 9.888 Untouched genes
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Reprinted from: Reboul et. al., Nat.Genet. 27, 332 (2001)
PCR amplification of C. elegans ORFsPCR amplification of C. elegans ORFs
PCR products of identified genes
PCR products of Untouched genes
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Reprinted from: Reboul et. al., Nat.Genet. 27, 332 (2001)
Successful PCR for the ORFs Successful PCR for the ORFs analyzedanalyzed
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Reprinted from: Reboul et. al., Nat.Genet. 27, 332 (2001)
ConclusionsConclusions
¤ 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.
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C. elegansC. elegans ORFeome ORFeome Version 3.1Version 3.1
Reprinted from: Lamesch et. al., Genome Research 14:2064-2069 (2004)
Gene prediction improvements
Classification of the 4232 repredicted and new ORFs
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The The C. elegansC. elegans ORFeome is an evolving ORFeome is an evolving resource resource
Reprinted from: Lamesch et. al., Genome Research 14:2064-2069 (2004)
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ConclusionsConclusions
¤ 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
Reprinted from: Lamesch et. al., Genome Research 14:2064-2069 (2004)
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Versatile Gene-Specific Sequence Tags for Versatile Gene-Specific Sequence Tags for ArabidopsisArabidopsis Functional Genomics Functional Genomics
¤ 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
Hilson et. al., Genome Research 14:2176-2189 (2004)
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Graphical representation of GSTsGraphical representation of GSTs
Reprinted from: Hilson et. al., Genome Research 14:2176-2189 (2004)
GSTPredicted gene
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GST productionGST production
Reprinted from: Hilson et. al., Genome Research 14:2176-2189 (2004)
High throughput PCR
High throughput verification
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GST cloning in GATEWAY vectors GST cloning in GATEWAY vectors
Reprinted from: Hilson et. al., Genome Research 14:2176-2189 (2004)
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The The Caenorhabditis elegansCaenorhabditis elegans PromoteromePromoterome
¤ 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
Dupuy et. al., Genome Research 14:2169-2175 (2004)
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The The C. elegansC. elegans promoterome 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
Reprinted from: Dupuy et. al., Genome Research 14:2169-2175 (2004)
ORF
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Overview of promoterome cloning Overview of promoterome cloning procedure procedure
Reprinted from: Dupuy et. al., Genome Research 14:2169-2175 (2004)
analysis of PCR products
large-scale cloning of the promoterome
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ConclusionConclusion
¤ 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 14:2169-2175 (2004)
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Applications of recombinational Applications of recombinational cloning cloning
Reprinted from: Dupuy et. al., Genome Research
14:2169-2175 (2004)
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Recommended readingRecommended 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 14:2176-2189 (2004)
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Further readingFurther reading
¤ ORFeome analysis– GATEWAY Recombinational Cloning
• Hartley et. al., Genome Research 10, 1788-1795 (2000) • Cheo et. al., Genome Research 14:2111-2120 (2004)• Walhout et al, Science 287: 116 (2000)
– C. elegans ORFeome• Lamesch et. al., Genome Research 14:2064-2069 (2004)
– C. elegans Promoterome• Dupuy et. al., Genome Research 14:2169-2175 (2004)