Functional genomics - medical.mums.ac.ir

Functional genomicsFunctional genomicsFunctional genomicsFunctional genomics

Completed genome projects

Eukaryotes: 23 complete; 242 assembly, 224 in progressViruses: 2 129 completeViruses: 2,129 complete

Bacteria: 714 complete

Archaea: 52 complete

Organellar: 1,709 complete

Metagenomics projects: 195

Why We Need Functional GenomicsWhy We Need Functional Genomics

Organism # genes% of genes with inferred function

Completion date of genomeOrganism # genes

E. coli 4288 60 1997yeast 6 600 40 1996yeast 6,600 40 1996C. elegans 19,000 40 1998Drosophila 12-14K 25 1999Arabadopsis 25,000 40 2000p ,mouse ~30,000? 10-20 2002h 30 000? 10 20 2000human ~30,000? 10-20 2000

Functional genomics in yeast (Jef Boeke)Functional genomics in yeast (Jef Boeke)

Protein level (proteom)Protein level (proteom)Two-hybrid screensAffinity purification and mass spectrometryPathwaysy

RNA level (transcriptom)MicroarraysSAGEtransposon tagging

Gene level (genome)Genetic footprintingTransposon insertion: random mutagenesisG d l ti t t d d l ti f ll ORF !!!Gene deletion: targeted deletion of all ORFs!!!

RNA levelRNA levelRNA levelRNA level

Subtracted cDNA LibrariesSubtracted cDNA LibrariesDifferential DisplayRepresentational Difference AnalysisSuppression Subtractive HybridizationSuppression Subtractive HybridizationcDNA MicroarraysSerial Analysis of Gene Expression2 D Gel Electrophoresis 2-D Gel Electrophoresis

Functional Functional GenomicsGenomicsDifferential Gene expression◦ cDNA-AFLP

◦ SAGESAGE◦ RDA/SSH

Identifying the Function of GenesF i l C l i◦ Functional Complementation◦ RNA interference/RNA silencing

DisclaimerDisclaimerDisclaimerDisclaimer

Relevant primarily to eukaryotesRelevant primarily to eukaryotesMost common systems (literature/class)Personal experience with themI like themI like them

mRNA abundance mRNA abundance classesclassesmRNA abundance mRNA abundance classesclassesSuperabundantSuperabundant◦ 15-90% of mRNA mass◦ <10 structural gene transcripts10 structural gene transcripts◦ >5000 molecules per cell per sequenceAbundant◦ 50-75% of mRNA mass◦ ~200-1000 structural gene transcripts (5% of diversity)

500 2500 l l ll ◦ 500-2500 molecules per cell per sequenceRare/complex◦ <25% of mRNA mass; individual seqs <0 01%◦ <25% of mRNA mass; individual seqs <0.01%◦ 95% of mRNA diversity◦ 1-10 molecules per cell per sequencep p q

SAGESAGESAGESAGE

Described by Velculescu et al (1995)Described by Velculescu et al. (1995)Originally 9 bp tags, now LongSAGE 21 bp10-50 tags in a cloneOnly requires a sequencer (and some Only requires a sequencer (and some time)

What is SAGE?What is SAGE?What is SAGE?What is SAGE?

Serial Analysis of Gene ExpressionSerial Analysis of Gene ExpressionMethod to quantify gene expression levels in samples of cellsOpen system Open system ◦ Can potentially reveal expression levels of all

genes: “unbiased” and “comprehensive”genes: unbiased and comprehensive◦ Microarrays are closed, since they only tell

b h d h you about the genes spotted on the array

How does SAGE work?How does SAGE work?How does SAGE work?How does SAGE work?

1 Isolate mRNA1. Isolate mRNA.

2. Bind RNA sample to oligo (dT) magnetic beads. The beads capture poly A+ RNA directly from your sample Synthesize double stranded cDNA onA+ RNA directly from your sample. Synthesize double stranded cDNA on the beads containing your mRNA using SuperScript. II reverse transcriptase and E. coli DNA polymerase

ACATGACTGACAGTTTGCTACATGATTGAGCCGTACATCCCAAAAAAAAATGTACTGACTGTCAAACGATGTACTAACTCGGCATGTAGGGTTTTTTTTTTTACATGACTGACAGTTTGCTACATGATTGAGCCGTACATCCCAAAAAAAAATGTACTGACTGTCAAACGATGTACTAACTCGGCATGTAGGGTTTTTTTTTTT

3. Digest the double stranded cDNA with a sequence specific restriction enzyme (an anchoring enzyme) that cleaves most transcripts at least once. Nla III is used as an anchoring enzyme since Nla III sites are known to occur approximately every 250 bp.

CATGATTGAGCCGTACATCCCAAAAAAAAATAACTCGGCATGTAGGGTTTTTTTTTTT

CATGATTGAGCCGTACATCCCAAAAAAAAATAACTCGGCATGTAGGGTTTTTTTTTTT

4. Divide the cDNA into two fractions and ligate with two adapters (A and B, 40 bp each) The adapters contain cohesive 4 bp overhangs complementary~40 bp each). The adapters contain cohesive 4-bp overhangs complementary

to the Nla III digested cDNA, a Type IIS restriction enzyme (tagging enzyme) recognition site at the 32 end, and priming sites for PCR amplification.

C A T G A T T G A G C C G T A C A T C C C A A A A A A A A AT A A C T C G G C A T G T A G G G T T T T T T T T T T T

G A T C G A T C G A T CC T A G C T A G C T A G G T A C





G A T C G A T C G A T CC T A G C T A G C T A G G T A CA







G A T C G A T C G A T CC T A G C T A G C T A G G T A CB

5. Cleave with Type IIS restriction enzyme (tagging enzyme), BsmF I. The yp y ( gg g y ),tagging enzyme binds to the recognition sequence in the adapter and cleaves the cDNA 10-14 bp downstream from the recognition site releasing a ~50-bp tag with a 4-bp overhang at the 52 end. The tag consists of ~40 bp of adapter sequence and 10-14 bp of unique sequence from a single transcript.

A d tA d tA d t

C A T GT A A C T C G G C A


A d a p te r





A d a p te rA d a p te r

T A A C T C G G C AC T A G C T A G C T A G G T A C

S A G E -ta g

T A A C T C G G C AC T A G C T A G C T A G G T A C T A A C T C G G C AC T A G C T A G C T A G G T A C

S A G E -ta gS A G E -ta gS A G E -ta g

6. Perform a Klenow reaction to fill-in the 5′ overhangs created by BsmF I digestion and ligate the t o fractions of tags to form 100 bp ditagsand ligate the two fractions of tags to form ~100-bp ditags.

C A T G A T T G A G C C G TT A A C T C G G C A






S A G E - ta gS A G E - ta gS A G E - ta g

GATCGATCGATCCATGATTGAGCCGTTGACTGATGGCATGGCGCATTGTTGTCTAGCTAGCTAGGTACTAACTCGGCAACTGACTACCGTACCGCGTAACAACAGATCGATCGATCCATGATTGAGCCGTTGACTGATGGCATGGCGCATTGTTGTCTAGCTAGCTAGGTACTAACTCGGCAACTGACTACCGTACCGCGTAACAACACTAGCTAGCTAGGTACTAACTCGGCAACTGACTACCGTACCGCGTAACAACACTAGCTAGCTAGGTACTAACTCGGCAACTGACTACCGTACCGCGTAACAACA

7 Amplify the ~100-bp ditags using primers specific for the primer binding sites in7. Amplify the 100 bp ditags using primers specific for the primer binding sites in the adapters to produce sufficient ditags for subsequent generation of concatemers. G.

GATCGATCGATCCATGATTGAGCCGTTGACTGATGGCATGGCGCATTGTTGTCTAGCTAGCTAGGTACTAACTCGGCAACTGACTACCGTACCGCGTAACAACAGATCGATCGATCCATGATTGAGCCGTTGACTGATGGCATGGCGCATTGTTGTCTAGCTAGCTAGGTACTAACTCGGCAACTGACTACCGTACCGCGTAACAACAGATCGATCGATCCATGATTGAGCCGTTGACTGATGGCATGGCGCATTGTTGTCTAGCTAGCTAGGTACTAACTCGGCAACTGACTACCGTACCGCGTAACAACA

102 bplinker-ditag

9 Ligate the 26-bp ditags to form concatemers Gel purify fractions containing 20-509. Ligate the 26 bp ditags to form concatemers. Gel purify fractions containing 20 50 tags/concatemer.

C G G GCCG G C G GG C G G GCCG G C G GGC G G GCCG G C G GG C G G GCCG G C G GGCATGATTGAGCCGTTGACTGATGGTAACTCGGCAACTGACTACCGTAC

CATGATTGAGCCGTTGACTGATGGTAACTCGGCAACTGACTACCGTAC

SAGE-tag SAGE-tagSAGE tag SAGE tag



SAGE-tagSAGE-tag SAGE-tagSAGE-tagSAGE tagSAGE tag SAGE tagSAGE tagg gSAGE-tag SAGE-taggg ggSAGE-tagSAGE-tag SAGE-tagSAGE-tag

> 500 bp

10. Clone the concatemers into the pZErO®-1 vector to obtain a SAGE. library. Seq ence selected clones Each transcript is identified b its niq e 10 14 bpSequence selected clones. Each transcript is identified by its unique 10-14-bp sequence tag and is quantified by the number of times the tag occurs within a given population of clones.

11 Sequencing the SAGE library11. Sequencing the SAGE library

FROM TAGS TO GENES……FROM TAGS TO GENES……Collect sequence records from GenBankCollect sequence records from GenBankAssign sequence orientation (by finding poly-A tail

l A i l f i )or poly-A signal or from annotations)Extract 10-bases -adjacent to 3’-most CATGAssign UniGene identifier to each sequence with a SAGE tagRecord (for each tag-gene pair)◦ #sequences with this tag#sequences with this tag◦ #sequences in gene cluster with this tag

Maps available at http://www.ncbi.nlm.nih.gov/SAGE

DIFFERENTIAL GENE DIFFERENTIAL GENE EXPRESSION BY SAGEEXPRESSION BY SAGE

Identification of differentially expressed Identification of differentially expressed genes in samples from different physiological or pathological conditions.Application of many statistical methodspp y

Poisson approximationBayesian methodBayesian methodChi square test.

SAGE software searches GenBank for SAGE software searches GenBank for matches to each tagThi ll i 3 i f This allows assignment to 3 categories of tags:

◦ mRNAs derived from known genes ◦ anonymous mRNAs, also known as expressed sequence

tags (ESTs) ◦ mRNAs derived from currently unidentified genes

SAGE SAGE VSVS MICROARRAYMICROARRAYSAGE SAGE VSVS MICROARRAYMICROARRAY

SAGE – An open system which detects both known and unknown transcripts and genes.

COMPARISONCOMPARISONCOMPARISON……COMPARISON……

SAGE MICROARRAYSAGEDetects 3’ region of transcript Restriction

MICROARRAYTargets various regions of the transcript Basetranscript. Restriction

site is determining factor.

of the transcript.Basecomposition for specificity of

Collects sequence

p yhybridization.Fluorescent signals and Co ects seque ce

information and copy no.Sequencing error and

signal intensity.Labeling bias and noise q g

quantitation bias. signals.

Contd……Contd……

Features SAGE MicroarrayDetects unknown Yes NoDetects unknown

transcriptsYes No

Quantification Absolute measure Relative measureQSensitivity High ModerateSpecificity Moderate High

Reproducibility Good for higher abundance transcripts

Good for data from intra-platform comparisoncomparison

Direct cost 5-10X higher than arrays.

5-10 X lower than SAGE

RECENT SAGE APPLICATIONS

•Analysis of yeast transcriptome•Gene Expression Profiles in Normal and Cancer Cellp•Insights into p53-mediated apoptosis•Identification and classification of p53-regulated genes•Analysis of human transcriptomesy p•Serial microanalysis of renal transcriptomes•Genes Expressed in Human Tumor Endothelium•Analysis of colorectal metastases (PRL-3)•Characterization of gene expression in colorectal adenomas and cancer•Using the transcriptome to analyze the genome (Long SAGE)

LIMITATIONS• Does not measure the actual expression level of a gene• Does not measure the actual expression level of a gene.

• Average size of a tag produced during SAGE analysis is ten bases and thismakes it difficult to assign a tag to a specific transcript with accuracy

• Two different genes could have the same tag and the same gene that isalternatively spliced could have different tags at the 3' ends

• Assigning each tag to an mRNA transcript could be made even more difficultand ambiguous if sequencing errors are also introduced in the process

• Quantitation bias:• Contamination of large quantities of linker-dimer molecules.• low efficiency in blunt end ligation.• Amplification bias.p

• Depending upon anchoring enzyme and tagging enzyme used, some fraction of mRNA species would be lost.

Advances over SAGEAdvances over SAGEAdvances over SAGEAdvances over SAGE

•Generation of longer 3` cDNA from SAGE tags f id tifi ti (GLGI)for gene identification (GLGI)

• Long SAGEg

• Cap Analysis of Gene Expression (CAGE)

• Gene Identification Signature (GIS)

S SAGE• SuperSAGE

• Digital karyotypingg y yp g

• Paired-end ditag

Long SAGELong SAGELong SAGELong SAGE

Increased specificity of SAGE tags for Increased specificity of SAGE tags for transcript identification and SAGE tag mapping.Collects tags of 21bpg pDifferent TypeII restriction enzyme-MmelAd SAGE l DNAAdapts SAGE principle to genomic DNA.Allows localisation of TIS and PAS.

CAGE CAGE (C d A l i f G E i )(C d A l i f G E i )CAGE CAGE (Capped Analysis of Gene Expression)(Capped Analysis of Gene Expression)

Aims to identify promotersAims to identify promoters.Collects 21 bp from 5’ ends of cap purified cDNA.U d i d h i diUsed in mouse and human transcriptome studies.The method essentially uses full-length DNA t th 5’ d f hi h li kcDNAs , to the 5’ ends of which linkers are

attached. Thi i f ll d b th l f th fi t 20This is followed by the cleavage of the first 20base pairs by class II restriction enzymes, PCR concatamerization and cloning of thePCR, concatamerization, and cloning of the CAGE tags

Reverse transcriptionAAAAA

AAAAABiotin•Full strand DNA synthesis•ssDNA release

Reverse transcription

Biotin + •ssDNA capture •Second strand synthesis

•ssDNA release

Mmel

xBiotin MmeI digestion of dsDNA

•Second strand synthesis

+

Xma JI

Biotin

Biotin Mmel PCR

Ligation to second linker

Mmel-PCR

Biotin

PCR amplification

Uni-PCR

X JI 1 2 X JI •Concatenation•Cloning XmaJI tag1 tag2 XmaJI •Cloning •Sequencing

Micro SAGEMicro SAGEMicro SAGEMicro SAGERequires 500-5000 fold less starting input RNARequires 500-5000 fold less starting input RNA.Simplifies by the incorporation of a ‘one tube’ procedure for all steps.procedure for all steps.Characterization of expression profiles in tissue biopsies, tumor metastases or in cases where tissue is p ,scarce. Generation of region-specific expression profiles of complex heterogeneous tissues.Limited number of additional PCR cycles are performed to generate sufficient ditag.

An expression profile can be obtained from as p plittle as 1-5 ng of mRNA.Comparison between the twoComparison between the two…

SAGE MicroSAGEAmount of input

material2.5-5 ug RNA 1-5 ng of mRNA

Capture of Streptavidin coated Streptavidin coated PCRCapture of cDNA

Streptavidin coated magnetic beads

Streptavidin coated PCR tube

Multiple tube vs. Subsequent reactions in Single tube reactionSingle tube

reactionmultiple tubes

Multiple PCI extraction and ethanol precipitation

Easy change of buffersNo PCI extraction or

ethanol ppt step.steps

pp pFewer manipulations

PCR 25-28 cycles 28 cycles followed by re-PCR 25 28 cycles 28 cycles followed by rePCR on excised ditag (8-

15)

SuperSAGESuperSAGESuperSAGESuperSAGE

Increases the specificity of SAGE tags and Increases the specificity of SAGE tags and use of tags as microarray probes.Type III RE EcoP15I – tag releasingCollects 26 bp tagsCollects 26 bp tagsHas been used in plant SAGE studies.Study of gene expression in which sequence information is not available.q

Flowchart of superSAGEFlowchart of superSAGE

Gene Identification Signature (GIS)Gene Identification Signature (GIS)Gene Identification Signature (GIS)Gene Identification Signature (GIS)

Identifies gene boundariesIdentifies gene boundaries.Collects 20bp LongSAGE tags from 3’ and 5’ end of the transcript.Applied to human and mouse Applied to human and mouse transcription studies.

DIGITAL KARYOTYPINGDIGITAL KARYOTYPINGDIGITAL KARYOTYPINGDIGITAL KARYOTYPING

Analyses gene structureAnalyses gene structure.Identification amplification and deletion in several cancerscancers.

PAIRED END DITAGPAIRED END DITAG

Identifies protein binding sites in genome.Applied to identify p-53 binding sites in the human genome.

11. SAGE: A LOOKING GLASS . SAGE: A LOOKING GLASS FOR CANCERFOR CANCER

Deciphering pathways involved in tumor genesis and identifying Deciphering pathways involved in tumor genesis and identifying novel diagnostic tools, prognostic markers,and potential therapeutic targets.

SAGE is one of the techniques used in the National Cancer Institute–funded Cancer GenomeAnatomy Project (CGAP). y j ( )

A database with archived SAGE tag counts and on-line query toolswas created - the largest source of public SAGE data.

More than 3 million tags from 88 different libraries have been gdeposited on the National Center for BiotechnologyEducation/CGAP SAGEmap web site (http://www ncbi nlm nih gov/SAGE/)(http://www.ncbi.nlm.nih.gov/SAGE/).

Several interesting patterns have emerged. g p g◦ cancerous and normal cells derived from the same tissue type are very

similar.◦ tumors of the same tissue of origin but of different histological type or

grade have distinct gene expression patterns◦ cancer cells usually increase the expression of genes associated with

proliferation and survival and decrease the expression of genes involved indifferentiationdifferentiation.

SAGE studies have been performed in patients with colon, pancreatic, lung bladder ovarian and breast cancerslung, bladder, ovarian, and breast cancers.

SAGE experiments validated in multiple tumor and normal tissue pairs using a variety of approaches including Northern blot analysis pairs using a variety of approaches, including Northern blot analysis, real-time PCR, mRNA in situ hybridization, andimmunohistochemistry.

Identification of an ideal tumor marker. E.g. Matrix metalloprotease1 in ovarian cancer is overexpressed.

22 IMMUNOLOGICAL STUDIES IMMUNOLOGICAL STUDIES22. IMMUNOLOGICAL STUDIES. IMMUNOLOGICAL STUDIESOnly a few SAGE analysis has been applied for the study of y y pp yimmunological phenomena.

SAGE analyses were conducted for human monocytes and their SAGE analyses were conducted for human monocytes and their differentiated descendants, macrophages and dendritic cells.

DC cDNA library represented more than 17,000 different genes. Genes differentially expressed were those encoding proteins related to cell motility and structure.

SAGE has been applied to B cell lymphomas to analyze genes involved in BCR –mediated apoptosis - polyamine regulation is involved in BCR mediated apoptosis. polyamine regulation is involved in apoptosis during B cell clonal deletion.

C tdC tdContd…Contd…LongSAGE has been used to identify genes of T cells with SLE that determine commitment to the disease.

Findings indicate that the immatureCD4+ T lymphocytes may be responsible for the pathogenesis of SLE.

SAGE has been used to analyze the expression profiles of Th-1 and Th-2 ll d l id tifi d f hi h i i 2 cells, and newly identified numerous genes for which expression is selective in either population.

Contributes to understanding of the molecular basis of Th1/Th2 dominated diseases and diagnosis of these diseases.

33 YEAST TRANSCRIPTOME YEAST TRANSCRIPTOME33. YEAST TRANSCRIPTOME. YEAST TRANSCRIPTOME

Yeast is widely used to clarify the biochemical Yeast is widely used to clarify the biochemical physiologic parameters underlying eukaryotic cellular functions.

Yeast chosen as a model organism to evaluate the power of SAGE technologypower of SAGE technology.

Most extensive SAGE profile was made for yeastMost extensive SAGE profile was made for yeast.

Analysis of yeast transcriptome affords a unique view of Analysis of yeast transcriptome affords a unique view of the RNA components defining cellular life.

44.ANALYSIS OF TISSUE .ANALYSIS OF TISSUE TRANSCRIPTOMESTRANSCRIPTOMES

Used to analyze the transcriptomes of renal cervical Used to analyze the transcriptomes of renal, cervical tissues etc.

Establishing a baseline of gene expression in normal tissue is key for identifying changes in cancer.y y g g

Specific gene expression profiles were obtained, and Spec c ge e e p ess o p o es we e obta e , a known markers (e.g., uromodulinin the thick ascending limb of Henle's loop and aquaporin-2 inthe collecting duct) were found.

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