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

Challenges Facing Genomics Challenges Facing Genomics D

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Detailed analysis of single gene

Genome sequencing

New tools in genomics:

microarrays and proteomics

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

Grow cells

Isolate RNAMake labeled cDNA

Mix and hybridize

Scan slideAnalyze data

Microarray experimental overview37C 25C

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 SP

• JH642 vs PY79 genomic DNA hybridization.– PY79 does not contain SP.– SP spots will be red.

JH642

PY79

Array size = 16mm x16mmSpot size = 150M

SP genes E. coli controlJH642

PY79