1. 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

2. Genes and Gene Expression Technology Display of Expression Information

3. Genomic DNA PromoterProtein coding sequenceTerminator What is a gene?

4. rRNA tRNA mRNA snRNA sRNA Genomic DNA Types of genes

5. TFIIB RNAPII TFIIA TFIID 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

6. TFIIB RNAPII TFIIA TFIID 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 repressors coactivators corepressors transcription apparatus chromatin factors RNA processing RNA transport RNA degradation

7. Activators Genome-wide Gene Expression (mRNA) can be Measured with DNA Microarrays Gene RNAPII TFIIH Transcription apparatus mRNA label hybridization ATGC TACG

9. Some Additional Numbers Yeast: 6200 genes ~200 transcriptional regulators Human: 30,000 - 50,000 genes ~1700 transcriptional regulators >100 cell types

10. Genes and Gene Expression Technology Display of Expression Information

11. Coated glass microarrays In situ oligonucleotide synthesis arrays DNA array technology

12. Hybridization and Scanning—Glass slide arrays - Prepare Cy3, Cy5- labeled ss cDNA - Hybridize 600 ng of labeled ss cDNA to glass slide array - Scan

13. 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

14. Cartesian PixSys 5500 with quill 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

15. Array Scanning Laser based - fluorescent emission

16. Spot Finding

18. 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

19. Rosetta Ink-jet printing Non-proprietary phosphoamidite oligonucleotide synthesis 29,000 oligos/1X3 inches 40-60 nucleotides/oligo More information: Hughes et al., Nature Biotechnology 2001

20. Genes and Gene Expression Technology Display of Expression Information

21. alpha 0’ alpha 7’ alpha 14’ alpha 21’ alpha 28’ alpha 35’ alpha 42’ alpha 49’ alpha 56’ alpha 63’ alpha 70’ alpha 77’ alpha 84’ alpha 91’ alpha 98’ alpha 105’ alpha 112’ alpha 119’ Mbp1/Swi6 Swi4/Swi6 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

22. ROS response

23. 600 Conditions/Mutations 6200 Genes Single-gene MutationsEnvironment Exercising the Genome

24. Visualization: Relative vs. absolute expression