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• 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

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Chromosome colored by dna2 left, wild type right

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dna2 left, wild type right

Chromosomes: red= high in mutant Graph:Left: mutant, rt: WT

blue=low in mutant, grey= null Color as in left side legend

WT and dna2-1 GeneSpring Data Presentations

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RelativeIntensity (ratio)

dna2 left, wild type right Selected : YBR060C (ORC2)

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

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dna2 left, wild type right Selected : YHR164C (DNA2)

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

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

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).

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

Microarray

Enlarged spots showing green (WT), red (mutant) and yellow

(about equal)

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

R/G = G/R = contents G Bg R Br R-Br/G-Bg G-Bg/R-Br

EMPTY 248 210 240 233 5.4285 0.184211

EMPTY 154 166 119 121 6.0000 0.166667 EMPTY 211 217 193 194 6.0000 0.166667 EMPTY 63 69 82 83 6.0000 0.166667 EMPTY 143 130 128 126 6.5000 0.153846 EMPTY 210 217 172 173 7.0000 0.142857 EMPTY 97 104 81 82 7.0000 0.142857 EMPTY 196 174 129 126 7.3333 0.136364 EMPTY 106 92 121 120 14.0000 0.071429 EMPTY 183 160 192 193 -23.0000 -0.04348 EMPTY 411 421 407 406 -10.0000 -0.1

Empty Spot “Hybridization”

Strains: Wild type (red), pol2-12 SIR3xtra (green)

RED GREEN r/g = g/r = Gene ID Red bk Grn bk R-bk/ G-bk/ Effect medians medians G-bk R-bk on: DNA Polymerases double mutant POL1 YNL102W 1178 921 1455 906 POL1 0.47 2.14 POL1 YNL102W 1536 1177 2054 1305 POL1 0.48 2.09 lower POL1 YNL102W 2417 303 2969 326 POL1 0.80 1.25 POL12 YBL035C 1311 294 2625 317 POL12 0.44 2.27 lower POL2 YNL262W 1802 286 3056 319 POL2 0.55 1.81 lower (predicted) POL2 YNL262W 1265 973 1572 1079 POL2 0.59 1.69 POL30 YBR088C 4923 661 7832 742 POL30 0.60 1.66 sl lower POL32 YJR043C 6346 1672 5756 1220 POL32 1.03 0.97 no signif change POL4 YCR014C 762 699 903 742 POL4 0.39 2.56 lower? POL5 YEL055C 1119 958 1059 896 POL5 0.99 1.01 prob not signif. POL5 YEL055C 578 1080 768 1025 POL5 1.95 0.51

Predefined Cluster: DNA Polymerases

Strains: Wild type (green), pol2-12 SIR3xtra (red)

Red Green R/G = G/R = Gene ID R br G bg R-br/ G-bg/ medians medians G-bg R-br

SIR1 YKR101W 1053 906 1076 981 SIR1 1.54736 0.64625 not signif. SIR2 YDL042C 1775 112 3841 124 SIR2 0.44740 2.23511 WT high SIR3 YLR442C 6216 174 949 231 SIR3 8.41504 0.11883 mut. high (pred.) SIR4 YDR227W 743 531 1076 546 SIR4 0.40000 2.50000 WT high (2 of 3) SIR4 YDR227W 797 693 924 718 SIR4 0.50485 1.98076 SIR4 YDR227W 1280 1123 1360 1137 SIR4 0.70403 1.42038

SIR3 Overexpression Affects Other Silent Information Regulators in the Same Complex

(Green is wild type mRNA; Red is mutant mRNA)

Double mutant: many cell cycle control genes are decreased in expression relative to wild type, even if they increased with extra dose of Sir3p alone. Jessica hypothesized: 1. The double mutant should have a slower cell cycle compared to wild type or single SIR3x mutant. It grew slower than the single SIR3x or wild type, but faster than pol2-12. 2. Phases of the cell cycle could be changed in the double mutant due to increased ability to deal with replication stalls or avoid checkpoint arrest.

WT/pol2-12 WT/SIR3x

WT/pol2-12SIR3x Role in cell:

gene R/G G/R R/G G/R R/G G/R CDC4 1.027 0.973 1.35 0.727 0.661 1.513 G1/S and G2/M CDC14 0.799 1.251 1.100 0.908 0.563 1.775 phosphatase, M exit & S CDC19 1.417 0.706 1.976 0.506 0.473 2.111 start (late G1) CDC20 0.773 1.292 1.697 0.589 0.439 2.276 APC (anaphase) CDC21 0.713 1.401 0.591 1.690 0.644 1.552 continuation of S CDC25 0.943 1.021 1.008 0.992 0.438 2.280 start control (signal td) CDC37 0.822 1.216 1.237 0.808 0.662 1.511 req. for passing start CDC47 1.011 0.989 1.287 0.777 0.384 2.603 initiation of S (helicase) CLB1 0.806 1.241 1.160 0.858 0.357 2.799 G2/M cyclin-dep. PK CLN1 0.823 1.213 0.983 1.017 0.506 1.975 G1/S cyclin-dep PK

Cell Cycle Control Gene Expression Changes

Cell Cycle Stages for pol2-12, SIR3x, and the Double Mutant

MG2 G1

S

Stage WT pol2-12

SIR3x double mutant

G1/S 45 min 45 min 25 min 25 min

S 90 min 180 min

90 min 150 min

G2/M 45 min 45 min 15 min 75 min