Topic intro slides

More complete coverage of components involved in gene expression

More information on expression technologies-what would the ideal chip do?-limitations to expression technologies

Datasets that have been produced thus far

Genes and Gene ExpressionTechnologyDisplay of Expression Information

Genomic DNA

Promoter Protein coding sequence Terminator

What is a gene?

rRNAtRNAmRNAsnRNAsRNA

Genomic DNA

Types of genes

TFIIB

RNAPII

TFIIATFIID

TFIIH TFIIF

Mediator

TFIIE

Activators

How are Genes Regulated? DNA-binding Activators Are Key To Specific Gene Expression

Gene

Chromatin modification complexes

Transcription initiation apparatus

TFIIB

RNAPII

TFIIATFIID

TFIIH TFIIF

Mediator

TFIIE

Activators

How are Genes Regulated? DNA-binding activators are key, but there are additional factors

Gene

Chromatin modification complexes

Transcription initiation apparatus activators

repressorscoactivatorscorepressorstranscription apparatuschromatin factorsRNA processingRNA transportRNA degradation

Activators

Genome-wide Gene Expression (mRNA) can be Measured with DNA Microarrays

GeneRNAPIITFIIH

Transcription apparatus

mRNA

mRNAlabel

hybridization ATGC

TACG

Some Additional Numbers

Yeast:6200 genes~200 transcriptional regulators

Human:30,000 - 50,000 genes~1700 transcriptional regulators>100 cell types

Genes and Gene ExpressionTechnologyDisplay of Expression Information

Coated glass microarrays

In situ oligonucleotide synthesis arrays

DNA array technology

Hybridization and Scanning—Glass slide arrays

- Prepare Cy3, Cy5- labeled ss cDNA

- Hybridize 600 ng of labeled ss cDNA to glass slide array

- Scan

Glass Slide DNA Arrays

• Large DNA molecules, PCR products, oligonucleotides, and proteins can be arrayed

• Typically non-specific binding to glass or coated surfaces

• High quality coated slides commercially available

Cartesian PixSys 5500 withquill printing technology

• 3 x 1 inch microscope slides, specially coated with amino groups• 50 slides/ bed• Up to 32 pins (quills)• 10,000 spots/slide• Spots are 100-200 µm in diameter• Hybridization volumes: 20-100ul

Array Scanning

Laser based - fluorescent emission

Spot Finding

Affymetrix high density oligonucleotide arrays: S. cerevisiae• Oligonucleotides synthesized in situ using light- directed, solid phase combinatorial chemistry

• 6181 ORFs + controls for alignment and normalization

• each ORF: 20 perfect match 25-mers (PM)20 single base mismatch partners (MM)

• mRNA signal = average of differences (PM-MM)

• average number of ORF mRNAs detected = 5460

• dynamic range = 0.1 - 100 mRNA molecules/cell

Hybridization and Scanning—Affymetrix arrays

Biotin-labeled cRNA target #1

- hybridize 10-40 g of cRNA to Affymetrix array

- Wash and scan

Compare data sets #1 & #2

Biotin-labeled cRNA target #2

- Wash and scan

Rosetta

Ink-jet printingNon-proprietary phosphoamidite oligonucleotide synthesis29,000 oligos/1X3 inches40-60 nucleotides/oligo

More information: Hughes et al., Nature Biotechnology 2001

Genes and Gene ExpressionTechnologyDisplay of Expression Information

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Mcm1/Fkh2/Ndd1

Ace2, Swi5, Mcm1

1 3.5 >5

1/3 1 3

Yeast Cell Cycle Gene Expression Program

Spellman et al. and Cho et al., 1998

800 Genes

>9 >6 >3 1:1 >3 >6 >9Fold repression Fold inductionGenomic Reprogramming in Response to Oxidant010204060120minutes One-third of genome expression is transiently reprogrammed6218 genes

ROS response

600 Conditions/Mutations

620

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Single-gene MutationsEnvironment

Exercising the Genome

Visualization: Relative vs. absolute expression