Microarrays in Undergraduate Research

1
Microarrays in Undergraduate Research

• Group Project and Senior Thesis Experiments
• Laura L. Mays Hoopes
• Pomona College/GCAT

2
Group Projects in Genetic Regulation in Eukaryotes

• Replication mutant of yeast/WT (sample dna2)
• Students prepare RNA, cDNA
• Students hybridize with microarray
• After washing, slides go to Davidson to be
  scanned
• Data are FTPed to us
• Scanalyze to add grids and get absorbances
• Excel sorting by ratio of absorbances
  Cluster/Treeview and/or GeneSpring analysis

3
WT and dna2-1 GeneSpring Data Presentations
Chromosomes red high in
mutant GraphLeft mutant, rt WT bluelow in
mutant, grey null Color as in left side legend
4
GeneSpring Analysis of Replication mRNAs in
dna2-1 and Wild Type -white bar indicates
gene selected by hand (ORC2) -redder color
indicates higher expression in dna2-1
dna2-1 WT
Relative
dna2 left, wild type right
Selected YBR060C (ORC2)
Intensity (ratio)
4.80
4.60
4.40
4.20
4.00
3.80
3.60
3.40
3.20
3.00
2.80
2.60
5.00
2.40
2.20
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
Sample
0.00
1
2
5
GeneSpring Analysis of Replication mRNAs in
dna2-1 and Wild Type -white bar indicates gene
selected by hand (DNA2) -redder color indicates
higher expression in dna2-1 dna2-1 WT
6
Group project on E. coli gene expression

• Ryan Skophammer and Sumire Kawai studied E. coli
  gene expression in Microbial Genetics, with Len
  Seligman
• The effects of a homing endonuclease creating a
  double strand break in a plasmid DNA were studied
• Normal cells were compared to cells with ara
  induced expression of the homing endonuclease

7
Results of E. coli gene expression study

• Prepared 8.68 ug good quality RNA from normal
  cells and 7.48 ug from the cells expressing
  the endonuclease
• 1055 ORFs increased at least 2x in the
  endonuclease cells 318 decreased to 0.5 or
  less.
• Genes that increased expression included 6
  subunits of DNA Pol III, DNA ligase, UmuC and D
  from the SOS repair pathway, rec B and rec C
  (rec A increased to 1.5x)
• Genes that decreased expression included MutS
  from mismatch repair, DscB inhibiting cell
  division, and Fla E and K for flagellar proteins
  (a surprise).

8
Senior Thesis Project of Jessica C. Brown

• Testing hypothesized interaction between Sir3p,
  chromatin silencer protein, and DNA replication
  protein DNA Polymerase epsilons major subunit
  (encoded by POL2)
• Growth curves, life spans, chromatid adhesion
  assays, and microarray analysis
• Microarrays compared WT/pol2-12, WT/SIR3x, and
  WT/pol2-12 SIR3x
• After examining microarray data, tested
  additional hypothesis about changes in cell cycle
  phase durations

9
Microarray
10
Enlarged spots showing green (WT), red (mutant)
and yellow (about equal)
11
Data from Microarrays

• Empty spot hybridization data
• Predefined clusters DNA Polymerases
• SIR3 Overexpression Affects other Sir proteins in
  the same complex
• Cell Cycle Control Gene Expression Changed in
  Double Mutant

12
Empty Spot Hybridization
13
Predefined Cluster DNA Polymerases
14
SIR3 Overexpression Affects Other Silent
Information Regulators in the Same Complex
15
Cell Cycle Control Gene Expression Changes
16
Cell Cycle Stages for pol2-12, SIR3x, and the
Double Mutant
M
G1
G2
S