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Functional Genomics in Non-Model Organisms. What is Functional Genomics? Functional genomics refers to the development and application of global (genome-wide.

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Presentation on theme: "Functional Genomics in Non-Model Organisms. What is Functional Genomics? Functional genomics refers to the development and application of global (genome-wide."— Presentation transcript:

1 Functional Genomics in Non-Model Organisms

2 What is Functional Genomics? Functional genomics refers to the development and application of global (genome-wide or system-wide) experimental approaches to assess gene function by making use of the information and reagents provided by structural genomics. It is characterized by high-throughput or large-scale experimental methodologies combined with statistical or computational analysis of the results (Hieter and Boguski 1997) Functional genomics as a means of assessing phenotype differs from more classical approaches primarily with respect to the scale and automation of biological investigations. A classical investigation of gene expression might examine how the expression of a single gene varies with the development of an organism in vivo. Modern functional genomics approaches, however, would examine how 1,000 to 10,000 genes are expressed as a function of development. (UCDavis Genome Center)

3 Functional Genomics Hunt & Livesey (eds.) Subtracted cDNA Libraries Differential Display Representational Difference Analysis Suppression Subtractive Hybridization cDNA Microarrays Serial Analysis of Gene Expression 2-D Gel Electrophoresis

4 My View of Functional Genomics Differential Gene expression –SAGE/MPSS –RDA/SSH –*Open systems* Identifying the Function of Genes –Functional Complementation –RNA interference/RNA silencing

5 Disclaimer Relevant primarily to eukaryotes Most common systems (literature/class) Personal experience with them I like them

6 Why We Need Functional Genomics Organism# genes % of genes with inferred function Completion date of genome E. coli yeast6, C. elegans19, Drosophila12-14K Arabadopsis25, mouse~30,000? human~30,000?

7 My Two Cents (as expressed by Hieter & Boguski 97) Functional genomics will not replace the time- honored use of genetics, biochemistry, cell biology and structural studies in gaining a detailed understanding of biological mechanisms. The extent to which any functional genomics approach actually defines the function of a particular protein (or set of proteins) will vary depending on the method and gene involved.

8 mRNA abundance classes (Okamuro & Goldberg) Superabundant –15-90% of mRNA mass –<10 structural gene transcripts –>5000 molecules per cell per sequence Abundant –50-75% of mRNA mass –~ structural gene transcripts (5% of diversity) – molecules per cell per sequence Rare/complex –<25% of mRNA mass; individual seqs <0.01% –95% of mRNA diversity –1-10 molecules per cell per sequence

9 SAGE & MPSS Serial Analysis of Gene Expression Massively Parallel Signature Sequencing Start from mRNA (euks) Generate a short sequence tag (9-21 nt) for each mRNA ‘species’ in a cell

10 Restriction digest double-stranded cDNA with a 4-base cutter “anchoring enzyme”; bind to streptavidin coated beads GGATGCATGXXXXXXXXXX CCTACGTACXXXXXXXXXX AAAA TTTT AAAA TTTT GTAC AAAA TTTT AAAA TTTT GGATGCATGOOOOOOOOOO CCTACGTACOOOOOOOOOO GGATGCATGXXXXXXXXXXOOOOOOOOOOCATGCATCC CCTACGTACXXXXXXXXXXOOOOOOOOOOGTACGTAGG CATG GTAC 1 CATG GTAC 2 12 Generate cDNA primed with biotin-oligo(dT) Divide pool in half & ligate to different linkers (1 or 2), both of which have a restriction site for the “tagging enzyme” Restriction digest with a Type IIS restriction enzyme, which recognizes the linker sequences and cuts downstream in a sequence independent fashion; fill-in 5’ overhang to blunt ends. Blunt end ligate pool 1 to pool 2, and PCR amplify with primers specific to linker sequences 1 and 2 Restriction digest with same anchoring enzyme (above); concatenate ditags and ligate to cloning/sequencing vector 1 2 AAAA TTTT AAAA TTTT GTAC Tag 1 Tag 2 Ditag CATGXXXXXXXXXXOOOOOOOOOOCATGXXXXXXXXXXOOOOOOOOOOCATG GTACXXXXXXXXXXOOOOOOOOOOGTACXXXXXXXXXXOOOOOOOOOOGTAC---- Ditag Tag 1Tag 2Tag 3Tag 4

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13 SAGE Described by Velculescu et al. (1995) Originally 9 bp tags, now LongSAGE 21 bp tags in a clone Only requires a sequencer (and some time)

14 MPSS Proprietary technology; published 2000 Generates 17 nt “signature sequence” Collects >1,000,000 signatures per sample Requires 2 µg of mRNA and $$

15 What is significantly different? Ruijter et al Physiol. Genomics 11:37-44.

16 What is significantly different?

17 Planning SAGE experiments…

18 How many tags need to be sequenced?

19 Comparing 2 libraries…

20 MPSS - Alexandrium fundyense unique tags; 3172 different at p<0.001

21 Not every tag is a unique sequence Not every sequence has a unique tag Alternative splicing, >1 tag per gene No restriction site, no tags per gene Sequencing error (random, 0.7% for SAGE, Velculescu et al. 1995) Antisense transcripts

22 Tag Abundance Distribution

23 Expression Ratio

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25 RDA Initially used for DNA comparisons (Lisitsyn et al. 1993) Later modified for cDNA to reduce complexity (Hubank and Schatz 1994) May need >1 enzyme to cover all genes Should pick up transcript present at <=0.005% Time-intensive + a LOT of manipulation

26 Success with RDA DNA markers in ginbuna (Murakami et al. 2002) mRNA induced under hypoxia in tiger salamander (McKean et al. 2002) Rice & date palm 2002; oak 2001; tobacco 2000; pea & maize 1998; earliest 1996 No more recent refs

27 MPSS - Alexandrium fundyense unique tags; 3172 different at p<0.001

28 Tester cDNA with Adaptor 1 Driver cDNA (in excess) Tester cDNA with Adaptor 2 a b c d { a,b,c,d + e a b c d e no amplification linear amplification exponential amplification fill in the ends first hybridization all components denatured second hyb: mix, add freshly denatured driver; anneal add primers; PCR amplify

29 Efficacy of SSH… Ji et al BMC Genomics 3:12 Diatchenko et al. 1996; could detect as little as 0.001% target Critical factor is relative concentration of target in tester and driver populations Effective enrichment when: –Target present at >= 0.01% –Concentration ratio>= 5-fold

30 What this looks like 208 signatures at >=0.01%, >= 5-fold induction

31 Success with SSH Armbrust 1999, diatoms Lots of biomedical refs 2003 Xylella, Aspergillus, Dunaliella

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33 Post-translational gene silencing FungiNeurosporaquellingtransgenes PlantsPetunia, Nicotiana, Arabadopsis, rice, tomato, potato, etc. PTGS Co-suppression transgenes viruses Animals: Invertebrates C. elegans Drosophila Paramecium Planaria Hydra T. brucei RNAi Co-suppression RNAI RNAi dsRNA transgenes dsRNA Animals: Vertebrates Zebrafish mouse RNAi dsRNA

34 Kamath et al ,757 strains = 86% of predicted ORFs Looked for sterility or lethality(Nonv), slow growth (Gro) or defects (Vpep) 1,722 strains (10.3% had such phenotypes)

35 Genes involved in basic metabolism & cell maintenance are enriched for Nonv phenotype Genes involved in more complex ‘metazoan’ processes (signal transduction, transcriptional regulation) are enriched for Vpep phenotype Nonv phenotypes highly underrepresented on the X chromosome X chromosome is enriched for Vpep phenotypes

36 Basal functions of eukaryotes are shared : - lethal (Nonv) genes tended to be of ancient origin - ‘animal-specific’ genes tended to be non-lethal (Vpep) - almost no ‘worm-specific’ genes were lethal

37 Genes producing a defective phenotype are clustered : Nonv clustered in central regions, except: on the X chromosome, which is underenriched for Nonv phenotypes

38 Functional Complementation Often yeast, E. coli The goal of the SGDP is to generate as complete a set as possible of yeast deletion strains with the overall goal of assigning function to the ORFs through phenotypic analysis of the mutants. As of 01/03, 95% of the approx ORFs have been deleted; more than 20,000 strains are available from Research Genetics, Open Biosystems and the ATCC.

39 Functional Complementation Intramembrane cleaving proteases: Drosophila rhomboid complements the aarA of Providencia stuartii and vice versa (Gallio et al. 2002) Cyclophilin-RNA interacting proteins in Paramecium, conserved from yeast to humans (Krzywicka et al. 2001)

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