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Microarrays in Undergraduate Research

Microarrays in Undergraduate Research. Group Project and Senior Thesis Experiments Laura L. Mays Hoopes Pomona College/GCAT. Group Projects in Genetic Regulation in Eukaryotes. Replication mutant of yeast/WT (sample: dna2) Students prepare RNA, cDNA Students hybridize with microarray

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Microarrays in Undergraduate Research

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  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 FTP’ed 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 Graph:Left: mutant, rt: WT blue=low 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 dna2-1dna2-1 WT dna2 left, wild type right Relative 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 dna2-1dna2-1 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 epsilon’s 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

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