Download Laura Hoopes - Davidson College

Microarrays in Undergraduate
Research
Group Project and Senior Thesis
Experiments
Laura L. Mays Hoopes
Pomona College/GCAT
Group Projects in Genetic
Regulation in Eukaryotes
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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
WT and dna2-1 GeneSpring Data Presentations
Chromosomes: red= high in mutant Graph:Left: mutant, rt: WT
blue=low in mutant, grey= null
Chromosome
colored by dna2 left, wild type right
Color as in left side legend
Relative
5.00 Intensity (ratio)
dna2 left, wild type right
I
4.80
4.60
II
4.40
III
4.20
4.00
IV
3.80
V
3.60
3.40
VI
3.20
VII
3.00
2.80
VIII
2.60
IX
2.40
2.20
X
2.00
XI
1.80
1.60
XII
1.40
XIII
1.20
1.00
XIV
0.80
XV
0.60
0.40
XVI
0.20
mito
Base pair
0
500000
1000000
1500000
Sample
0.00
1
2
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
dna2 left, wild type right
Selected : YBR060C (ORC2)
Relative
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
2.40
5.00
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
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
5.00
dna2 left, wild type right
Selected : YHR164C (DNA2)
Relative
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
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
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
Empty Spot “Hybridization”
contents
G
EMPTY
EMPTY
EMPTY
EMPTY
EMPTY
EMPTY
EMPTY
EMPTY
EMPTY
EMPTY
EMPTY
248
154
211
63
143
210
97
196
106
183
411
Bg
210
166
217
69
130
217
104
174
92
160
421
R
240
119
193
82
128
172
81
129
121
192
407
Br
R/G =
G/R =
R-Br/G-Bg G-Bg/R-Br
233 5.4285 0.184211
121 6.0000 0.166667
194 6.0000 0.166667
83 6.0000 0.166667
126 6.5000 0.153846
173 7.0000 0.142857
82 7.0000 0.142857
126 7.3333 0.136364
120 14.0000 0.071429
193 -23.0000 -0.04348
406 -10.0000
-0.1
Predefined Cluster: DNA Polymerases
Strains: Wild type (red), pol2-12 SIR3xtra (green)
RED GREEN
Gene ID Red bk Grn bk
medians medians
r/g = g/r =
R-bk/ G-bk/ Effect
G-bk R-bk on:
DNA Polymerases
POL1 YNL102W 1178 921 1455 906 POL1 0.47
POL1 YNL102W 1536 1177 2054 1305 POL1 0.48
POL1 YNL102W 2417 303 2969 326 POL1 0.80
POL12 YBL035C 1311 294 2625 317 POL12 0.44
POL2 YNL262W 1802 286 3056 319 POL2 0.55
POL2 YNL262W 1265 973 1572 1079 POL2 0.59
POL30 YBR088C 4923 661 7832 742 POL30 0.60
POL32 YJR043C 6346 1672 5756 1220 POL32 1.03
POL4 YCR014C 762 699 903 742 POL4 0.39
POL5 YEL055C 1119 958 1059 896 POL5 0.99
POL5 YEL055C 578 1080 768 1025 POL5 1.95
double mutant
2.14
2.09 lower
1.25
2.27 lower
1.81 lower (predicted)
1.69
1.66 sl lower
0.97 no signif change
2.56 lower?
1.01 prob not signif.
0.51
SIR3 Overexpression Affects Other Silent
Information Regulators in the Same Complex
Strains: Wild type (green), pol2-12 SIR3xtra (red)
Gene ID
SIR1
SIR2
SIR3
SIR4
SIR4
SIR4
Red
Green
R br G bg
medians medians
R/G = G/R =
R-br/ G-bg/
G-bg R-br
YKR101W 1053 906 1076 981 SIR1 1.54736
YDL042C 1775 112 3841 124 SIR2 0.44740
YLR442C 6216 174 949 231 SIR3 8.41504
YDR227W 743 531 1076 546 SIR4 0.40000
YDR227W 797 693 924 718 SIR4 0.50485
YDR227W 1280 1123 1360 1137 SIR4 0.70403
0.64625
2.23511
0.11883
2.50000
1.98076
1.42038
not signif.
WT high
mut. high (pred.)
WT high (2 of 3)
Cell Cycle Control Gene Expression Changes
(Green is wild type mRNA; Red is mutant mRNA)
WT/pol2WT/pol212
WT/SIR3x 12SIR3x
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
CDC14 0.799 1.251 1.100 0.908 0.563 1.775
CDC19 1.417 0.706 1.976 0.506 0.473 2.111
CDC20 0.773 1.292 1.697 0.589 0.439 2.276
CDC21 0.713 1.401 0.591 1.690 0.644 1.552
CDC25 0.943 1.021 1.008 0.992 0.438 2.280
CDC37 0.822 1.216 1.237 0.808 0.662 1.511
CDC47 1.011 0.989 1.287 0.777 0.384 2.603
CLB1 0.806 1.241 1.160 0.858 0.357 2.799
CLN1 0.823 1.213 0.983 1.017 0.506 1.975
Role in cell:
G1/S and G2/M
phosphatase, M exit & S
start (late G1)
APC (anaphase)
continuation of S
start control (signal td)
req. for passing start
initiation of S (helicase)
G2/M cyclin-dep. PK
G1/S cyclin-dep PK
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.
Cell Cycle Stages for pol2-12, SIR3x, and the Double
Mutant
M
G1
G2
S
Stage
WT
pol212
SIR3x double
mutant
G1/S
45 min 45 min 25 min 25 min
S
90 min 180
min
G2/M
45 min 45 min 15 min 75 min
90 min 150
min