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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. 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 • 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. 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. 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). 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. 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. Quantitative real time RT-PCR Depth of knowledge Challenges Facing Genomics Detailed analysis of single gene New tools in genomics: microarrays and proteomics Genome sequencing Breadth of knowledge 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 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) 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. 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 Microarray experimental overview 37C 25C Grow cells Isolate RNA Make labeled cDNA Mix and hybridize Scan slide Analyze data Hybridization: basic concept The ability of two strands to hybridize is dependent on their complementarity. More complementarity=better hybridization Bacterial DNA microarrays • • • • Small genome size Fully sequenced genomes, well annotated Ease of producing biological replicates Genetics 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. 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 SPb. • JH642 vs PY79 genomic DNA hybridization. – PY79 does not contain SPb. – SPb spots will be red. JH642 PY79 Array size = 16mm x16mm Spot size = 150mM JH642 PY79 SPb genes E. coli control