Formation of a Consortium for Teaching Genomics: Genome Consortium for Active Teaching GCAT A. Malco

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Using DNA Microarrays from Multiple Species 
Comparisons for Teaching Effectiveness
Todd T. Eckdahl Biology Department Missouri
Western State College
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Overview

• Background
• Courses using microarray technology
• Species studied
• Implementation
• Results
• Planned research projects

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Missouri Western State College

• Saint Joseph, Missouri
• State-supported PUI
• 5200 students
• 200 faculty
• Biology Department
• 340 majors
• 10 faculty
• No graduate degree programs
• New major in Biochemistry and Molecular Biology
  

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Courses Using Microarray Technology

• BIO 431 Molecular Biology
• 4 credit course
• 3 hours lecture, 3 hours lab
• Student majors
• BMB, Biology with Health Sciences emphasis
• BIO 313 Topics in Molecular Genetics
• 1 credit course
• 3 hours lab
• Student majors
• BMB, Biology-Health Sciences, Teaching

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Functional Genomics Technology

• Microarrays
• cDNAs printed
• eg. Stanford yeast chips, UW E. coli chips
• 80-mer oligos printed
• eg. ISB yeast chips
• Labeling options
• Indirect labeling
• eg. Genisphere dendrimers
• Direct incorporation
• eg. Ulysis alexafluore labeling

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Conducting Microarray Experiments in a Course

• Emphasize the Big Picture
• Genomics, functional genomics, proteomics
• Shift to data-rich science
• Primary Literature
• Brainstorming for ideas
• Scheduling
• Data Analysis
• Presentations

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Ideas for Yeast Experiments

• Glucose vs. Galactose vs. Fructose vs. Maltose
• Anaerobic vs. aerobic
• Induction of sporulation
• Heat Shock v. Cold Shock
• Drug treatment

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Minor Groove Binding Drugs

• Anti-tumor properties
• Conformational change in the 3D structure of DNA
• Prior Knowledge of MGBD/DNA interaction
• As models for minor
• groove binding proteins

DAPI
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Yeast Culture

• OD at 660 nm to measure turbidity
• Grown through log phase
• 4 hours of exposure to 10 uM DAPI
• Control culture without DAPI

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Isolation of RNA

• Sterile, RNase- free equipment and workspace
• Harvesting of yeast
• Production of spheroblasts
• Isolation of RNA via RNA spin column
• Elution of RNA
• Quantify RNA with A260 / A280
• Run RNA on denaturing agarose

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Preparation of labeled cDNA and hybridization

• Reverse transcription of RNA
• capture sequence incorporated
• Label preparation
• addition of Cy3 and Cy5 dendrimer
• addition of capturing reagents
• Add probe to slide, cover and incubate at 55 C
  for 1-3 days

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Experimental Summary
Yeast in log phase
untreated
10 uM DAPI
Total RNA
Total RNA
Reverse Txn
cDNA
cDNA
Red fluorophore
Green fluorophore

microarray
hybridization
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Data Acquisition

• Post-hybe wash, dry slide
• Ship for scanning
• Receive data
• Scanalyze
• Submit to SMD

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Microarray Controls

• Empty or 3X SSC
• Duplicate genes
• Duplicate experiments
• Cy3 and Cy5 dyes
• Poly A
• Genomic, Intron, tRNA

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Example of induced gene
YBR012W-B, TyB Gag-Pol protein TGAGAAGCTGTCATCGA
AGTTAGAGGAAGCTGAAGTGCAAGGATTGATAA
TGTAATAGGATAATGAAACATATAAAACGGAATGAGGAATAATCGCAAT
A TTAGTATGTAGAAATATAGATTCCATTTTGAGGATTCCTATATCCT
CGAG GAGAACTTCTAGTATATTCTGTATACCTAATATTATAGCCTTT
ATCAACA ATG
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Example of repressed gene
YHR055C, copper-binding metallothionein TTCCGCTGA
ACCGTTCCAGCAAAAAAGACTACCAACGCAATATGGATTGT CAGAATC
ATATAAAAGAGAAGCAAATAACTCCTTGTCTTGTATCAATTGC ATTAT
AATATCTTCTTGTTAGTGCAATATCATATAGAAGTCATCGAAATA GAT
ATTAAGAAAAACAAACTGTACAATCAATCAATCAATCATCACATAAA A
TG
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Analyses at Stanford Microarray Database

• Single spot or sequence
• Data filtering
• signal strength
• R/G or G/R ratio
• Linear regression comparison
• Prepare data for clustering

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Databases linked to SMD

• SGD - Saccharomyces genome database
• Genbank
• YPD - yeast protein database
• Swissprot protein database

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Ideas for E. coli Experiments

• Metabolic shift
• Osmotic stress
• Growth curve effects
• Heat Shock v. Cold Shock
• Drug treatment
• Effects of gene deletion

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BIO 313 Experiment

• E coli chips
• M1655 sequenced strain
• cDNA spotted
• Putative transcriptional regulators
• nusA deletion strain
• yhbM deletion strain
• Two channel hybridizations
• Compare labeled RNA from wt versus deletion

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E. coli culture

• Overnight culture
• Grown at 37C to log phase
• OD at 600 nm to measure turbidity

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RNA Isolation

• Sterile, RNase-free equipment and work area
• Total RNA SafeKit
• Total RNA Safe protocol used
• Lysis of E. coli done with mixture of TE and
  lysozyme

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RNA Isolates

• Measure A260 / A280
• Check on denaturing
• agarose gel

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Labeling

• Labeling of isolated RNA done by use of ULYSIS
  Nucleic Acid Direct Labeling Kit
• ULYSIS protocol followed
• Fluorescent Dyes Alexa Fluor 546 (green) and
  Alexa Fluor 660 (red)

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Hybridization

• Microarray prehybridized
• Labeled RNA mixed together in hybridization
  buffer and added to slide
• Hyb at 55 C in dry incubator overnight
• Post-hyb washes

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Microarray Controls

• Empty or 3X SSC
• Duplicate genes
• Duplicate experiments
• Cy3 and Cy5 dyes
• Genomic

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Examples of Results
asr flhC emrY
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Examples

• Induced
• Asr, G1787881
• acid shock protein
• Repressed
• flhC, G1788201
• regulator of flagellar biosynthesis acting on
  class 2 operons
• Non-responsive
• emrY, G1788710
• multidrug resistance protein Y
• putative transport

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Example of induced gene
Asr, G1787881 gatca agactactattattggtagctaaatttccc
ttaagtcac aatacgttattatcaacgctgtaatttattcagcgtttg
tacatatcgttacacgctgaaaccaaccactcacggaag
tctgccattcccagggatatagttatttcaacggccccg
cagtggggttaaatgaaaaaacaaattgagggtatgaca 1 - atg
aaa aaa gta tta gct ctg gtt gtt gcc 31 - gct gct
atg ggt ctg tct tct gcc gcc ttt 61 - gct gca gag
act acg acc aca cct gct ccg 91 - act gcg acg acc
acc aaa gca gcg ccg gcg
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Example of repressed gene
flhC, G1788201 ccgca aatggttaagctggcagaaaccaatcaac
tggtttgtca cttccgttttgacagccaccagacgattactcagttgac
gcaagattcccgcgttgacgatctccagcaaattcatac
cggcatcatgctctcaacacgcttgctgaatgatgttaa
tcagcctgaagaagcgctgcgcaagaaaagggcctgatc 1 - atg
agt gaa aaa agc att gtt cag gaa gcg 31 - cgg gat
att cag ctg gca atg gaa ttg atc 61 - acc ctg ggc
gct cgt ttg cag atg ctg gaa 91 - agc gaa aca cag
tta agt cgc gga cgc ctg
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Example of non-responsive gene
emrY, G1788710 gaact catggaacaccccttgcgtattggtttat
cgatgacagc aactattgatacgaagaacgaagacattgccgagatgcc
tgagctggcttcaaccgtgacctccatgccggcttatac
cagtaaggctttagttatcgataccagtccgatagaaaa
agaaattagcaacattatttcgcataatggacaacttta 1 - atg
gca atc act aaa tca act ccg gca cca 31 - tta acc
ggt ggg acg tta tgg tgc gtc act 61 - att gca ttg
tca tta gcg aca ttt atg caa 91 - atg ttg gat tcc
act att tct aac gtc gca
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Microarrays in CoursesLessons Learned

• Advance planning essential
• Controls for critical steps
• Reliability and Reproducibility
• Do Controls Make Sense?
• Do Results Make Sense?
• Potential for large amounts of data means
  extensive analysis time needed

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Ongoing and Planned Research Projects

• Measure Effects of Minor Groove Binding Drugs on
  Gene Expression in Yeast
• Measure Effects of Minor Groove Binding Drugs on
  Gene Expression in Human Tumor Cells in Culture

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Big Ideas

• Sequence and structural requirements for MGBD
  binding
• AT rich sequences
• DNA bending
• Determination of optimal binding sites
• Effects of MGBDs on gene expression
• Preliminary data using RT-PCR
• Global patterns of gene expression
• Complementary in vitro and in vivo approaches

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Acknowledgements

• Genome Consortium for Active Teaching
• Malcolm Campbell, Davidson College
• NSF DBI 0099720 MUE grant
• Dr. Barbara Dunn, Stanford University
• Dr. Fred Blattner lab, UW-Madison
• Dr. Bob Getts, Genisphere, Inc.
• Missouri Western Students