1. Gene expression and DNA microarrays Old methods. New methods based on genome sequence. –DNA Microarrays Reading assignment - handout –Chapter 4 - 195-232, 239-250, 255 MIAME –Box 4.1, 4.2 (Significance test only), 4.3, and 4.4.

2. Genome of the week E. coli O157:H7. –Comparison to the laboratory strain of E. coli. Causes haemorrhagic colitis –Initially identified in 1982 during an outbreak of severe bloody diarrhea. –Linked to contaminated ground beef from Michigan –Can be lethal 75,000 cases per year

3. Major findings: –Comparison of E. coli O157:H7 with E. coli K-12 (common lab strain) found that the O157:H7 genome is ~ 1Mb larger than K-12 and contains 1,387 genes specific for O157:H7. –Genomes share a 4.1 Mb backbone with species specific DNA interspersed throughout the genome K-islands - specific to K-12 (0.53Mb) O-islands - specific –Lateral transfer of DNA occurs much more frequently than previously thought. Especially high for enterobacteria. O-island specific DNA encoded genes required for virulence and a large number of phage and phage associated genes. Five sequences of E. coli - 4 pathogens and 1 lab strain. –Pathogens are as different from each other as they are from the non-pathogenic lab strain.

4. Gene expression What is gene expression? Methods for measuring a single gene. –Northern Blots –Reporter genes –Quantitative RT-PCR Operons, regulons, and stimulons. DNA microarrays. –Expression profiling –Identifying protein binding sites. –Comparing gene content of different strains.

5. What is gene expression? The amount of RNA produced from a gene. Level of RNA produced from a gene is controlled by: –Transcription –Stability/Degradation Transcriptome - Expressed transcripts in a cell under defined experimental conditions. –mRNA(5-10% of total RNA). –rRNA, tRNA - make up most of total RNA –scRNA (protein secretion), tmRNA (rescue stalled ribosomes).

6. Regulons and Stimulons Operon - group of genes co-expressed on a single transcript. –One location of the genome Regulon - genes that are regulated by a single transcription factor. –Genes and operons throughout the genome Stimulon - collection of genes that are regulated in response to environmental changes. –Can be multiple regulons affected at once. Regulatory network - alternative term for regulon.

7. Analysis of gene expression at the single gene level. Northern Blots –Measure RNA levels by hybridization of a labeled probe to total RNA. Reporter Genes –Use of an enzyme to measure the amount of transcription from a promoter. Quantitative real-time RT-PCR. Brief review in book.

8. Quantitative real time RT-PCR

9. Challenges Facing Genomics Depth of knowledge Breadth of knowledge Detailed analysis of single gene Genome sequencing New tools in genomics: microarrays and proteomics

10. Assaying the regulation of 1000s of genes in a single experiment DNA microarrays –DNA molecules printed at high density used to determine the level of RNA or DNA in a sample. –Can be thought of a “reverse Northern blots” Other technologies (described in chapter 4). –SAGE –Microbeads

11. DNA Microarrays -Introduction Spotted DNA arrays (glass slides) –Competitive binding of samples - internal control –Fluorescent detection - Cy3 and Cy5 –Small sample sizes (10-30µl). –PCR or cDNA arrays - double stranded –Long oligonucleotide arrays - single stranded Better specificity, cheaper, easier to work with. Short oligonucleotide arrays –ex. Affymetrix DNA spotted onto nylon membranes (macroarrays)

12. Applications of DNA microarrays Expression profiling –Determining the relative levels of RNA in two or more samples. DNA/DNA hybridizations –Investigate gene content between different strains –Determine gene dosage –16S arrays - microbial communities (being developed). Identification of protein binding sites –ChIP-Chip. Immunoprecipitation of protein/DNA complexes. Assaying those interactions with microarrays.

13. Uses of DNA microarrays Detection of candidate genes –Expression profiling –DNA/DNA hybridizations Annotation of gene function –Expression - compendium approach Defining regulatory networks –Expression profiling –ChIP/chip experiments Molecular phenotyping –Expression profiling –DNA/DNA hybridizations

14. Microarray experimental overview 37C25C

15. Hybridization: basic concept The ability of two strands to hybridize is dependent on their complementarity. More complementarity=better hybridization

16. Bacterial DNA microarrays Small genome size Fully sequenced genomes, well annotated Ease of producing biological replicates Genetics

17. B. subtilis DNA microarrays PCR generated microarrays using custom primers (Sigma-Genosys). Each PCR product represents a single gene. 4074 genes of 4101 on the array. Printed on Corning CMT-GAPS slides. 4 E. coli controls, each represented 15-20 times on the array.

18. How a DNA microarray works Comparing the genome content of two B. subtilis strains. The two strains differ only by the fact that JH642 is lysogenized with the bacteriophage SP  JH642 vs PY79 genomic DNA hybridization. –PY79 does not contain SP . –SP  spots will be red.

19. JH642 PY79 Array size = 16mm x16mm Spot size = 150  M

20. SP  genes E. coli control JH642 PY79