ASU TVDC Progress Report
11/24/09
Kathryn F. Sykes and Stephen A. Johnston
Completed Milestones: 25, 26, 28 and 32, 33, 34
Active Milestones: ASU 35 &36
Currently Inactive Milestones: 30, 37, 38
Slide 1
Transcriptome Specific Aims
• Is there a relation between expression of genes in vivo
and antigenicity?
• Previously used in studies of Mycobacterium
tuberculosis and Yersinia pestis
• Using LAPT to identify gene expression patterns over
the early time course of infection with Francisella
tularensis Schu S4
Slide 2
Problems with Measuring
Bacterial mRNA In Vivo
- Low amount of bacterial mRNA
- No poly A tail
- Amount and complexity of host mRNA
Slide 3
LAPT Process
Slide 4
MILESTONE 35
Array hybridations with mouse RNAs
from virulent Schu 4 infection
& RT PCR confirmation of candidates
Gray: (sub )milestone title
Red: completed
Green: in progress
Virulent Schu 4 Samples
RT-PCR Confirmations
Initial samples
Dose-Response of Infection
Ongoing
Slide 5
MILESTONE 36
Final integration of expression data
and informatics analysis
Gray: (sub )milestone title
Red: completed
Green: in progress
Ongoing
Slide 6
Experimental Design
Sample Sets
High-Dose Mouse
Challenge
LAPT
2
1
1
Rat
2
1
1
2
1
1 2
1 2
2
2
1
2
Slides
1 2
1
2
1 2
1 2 1 2 1 2
Red = completed
Time Course = 1, 3, 5, 7, and 24 hours post challenge
Slide 7
Genomically-Normalized Signal Intensity
Pattern Mapping
Up
Flat
Down
1
3
5
7
24
Slide 8
Pattern Mapping
Create a pattern
Pearson Correlation
Identifies Matches
Pattern Mapping v 2
Identify a gene
FTT1357c
FTT1712c
FTT0901
FTT0904
FTT0721c
iglC
iglC1
lpnA
lpnB
katG
Pearson Correlation
Identifies Matches
Pattern Mapping
Comparison across Datasets
Mouse
Rat
Statistical Selection
Significance between Time Frames 1 vs 3 hour
Strategies
• Using pattern maps to identify the top ~200 genes in one
of three patterns
– Increasing; Decreasing; Flat
• Venn the individual experiment lists to get a restricted list
of genes
• Compare the restricted sets of genes between
experiments to identify those across experiments
• Fold Selections for narrowing final lists
• Mapping Literature-based gene lists on our data
Slide 13
Overlap of Pattern Map – Selected Gene Lists
Mouse Down
Rat
UP
Mouse
Rat
186
14
173
186
Mouse
104
Flat
2
27
172
Rat
89
Slide 14
Top Selections
Cross-Species Model – Pattern Maps
Increasing
FTT0145
FTT0351
FTT0415
FTT0583
FTT0609
FTT0680c
FTT0698
FTT1069c
FTT1119
FTT1137c
FTT1226c
FTT1489
FTT1502
FTT1623c
rpoC
rplQ
glgC
fopA
peptidase, M24 family protein
pth
rpsO
hypothetical protein
LysR transcriptional regulator family protein
hypothetical protein
iscS
hypothetical protein
aromatic amino acid HAAP transporter
hypothetical protein
Decreasing
FTT0169
FTT0195c
FTT0225c
FTT0229c
FTT0235c
FTT0314
FTT0477c
FTT0495
FTT0557
FTT0560c
FTT0798
FTT0871
FTT1038c
FTT1070c
FTT1115c
FTT1254
FTT1281c
FTT1288
FTT1307c
FTT1311
FTT1391
FTT1470c
FTT1506
FTT1513
FTT1727c
FTT1763c
FTT1771
HemK protein
L-glutaminase
pseudogene
Flat
FTT0114
FTT0150
FTT1526c
deoC
rpsP
idh
efp
rnpA
tsf
birA
hypothetical protein
AhpC/TSA family protein
serC
glycosyl transferase family protein
gpsA
rpsU
pseudogene
secD
fadD1
yhbH
hypothetical protein
hypothetical protein
yieO
panD
gmk
hypothetical protein
pseudogene
multidrug resistance protein, membrane located
acetyltransferase protein
hypothetical protein
Slide 15
Pattern Mapping with Fold Selection
Mouse
Fold Change 1 > 24
(Top 107 Up Over Time - .524)
(21 - 5 fold)
Pattern Mapping with Fold Selection
Mouse
Fold Change 1 > 24
(Top 200 Down Over Time - .839)
(21 - 10 fold)
Top Mouse Selections
Pattern Mapping with Fold Selection
Increasing
FTT0038
FTT0039
FTT0142
FTT0364c
FTT0427
FTT0498c
nuoH
nuoI
rplJ
hypothetical protein
thrB
pseudogene
FTT0506c hypothetical protein
FTT0512 glutamine amidotransferase subunit PdxT
FTT0535c mdh
glycerophosphoryl diester phosphodiesterase
FTT0726c family protein
FTT0753 rpsU
FTT0785 hypothetical protein
FTT0838 tolR
FTT1119 LysR transcriptional regulator family protein
FTT1137c hypothetical protein
FTT1336
FTT1446
FTT1570c
FTT1576
FTT1730c
cydC
rho
fabZ
hypothetical protein
amino acid transporter
FTT1801c trpG1
Decreasing
FTT0216
FTT0473
FTT0560c
FTT0815c
FTT0840
FTT0876c
Flat
isftu1
accC
serC
hypothetical protein
tolB
aroC
beta-glucosidase-related
FTT0928c glycosidase
FTT0961 mdaB
FTT0977c hypothetical protein
FTT0022
FTT0023c
FTT0044
FTT0069c
FTT0099
FTT0101
FTT1010
FTT1305c
FTT1355
FTT1391
FTT1418c
FTT1448c
helix-turn-helix family protein
murA
hypothetical protein
panD
nusB
manC
FTT0117
FTT0226c
FTT0497c
FTT0519
FTT0630
FTT0683c
FTT1450c
FTT1454c
FTT1506
FTT1695
FTT1725c
wbtM
wbtJ
hypothetical protein
groES, htpA
pcm
FTT1760
nhaA
hypothetical protein
lipase/acyltransferase
nuoN
hypothetical protein
isftu2
hypothetical protein
FTT0103c hypothetical protein
FTT0106c RND efflux transporter
FTT0107c dsbB
tmk
isftu1
pseudogene
NIF3 family protein
hfq
pilD
iron-sulfur cluster insertion
FTT0700 protein ErpA
FTT0914c lspA
FTT1438c pseudogene
FTT1718c isftu1
FTT1779 pseudogene
Slide 18
Pattern Mapping of Wehrly et al Datasets
From 37 Upregulated genes
Mapped on Mouse
2 fold Change 1 < 24
FTT0625
FTT0113
FTT0626
FTT1124
clpX
deoB
lon
metN
Pattern Mapping of Wehrly et al Datasets
From 10 Downregulated genes
Mapped on Mouse
2 fold Change 3 > 24
FTT0051
FTT0584
FTT0154
FTT1471c
FTT0806
FTT1442c
rbfA
xerD
deaD
capC
rpoA2
Pattern Mapping of Wehrly et al Datasets
3 Knock-outs
Mapped on Mouse
FTT0383
FTT0369c
FTT1676
hypothetical protein
hypothetical protein
hypothetical protein
Pattern Mapping of Literature-Based Gene Lists
FPI-1 (FTT1344-1360c)
Pattern Mapping of Literature-Based Gene Lists
General Stress Response Genes
FTT0356
FTT0624
FTT0625
FTT0626
FTT0687c
FTT0688c
FTT1268c
FTT1269c
FTT1270c
FTT1512c
FTT1695
FTT1696
FTT1769c
FTT1794
HtpG
ClpP
ClpX
Lon
HslU
HslV
dnaJ
dnaK
GrpE
DnaJ1
groES, htpA
groEL, mopA
ClpB
small HSP
Slide 23
Pattern Mapping of Literature-Based Gene Lists
Oxidative Stress Response Genes
FTT0086
FTT0259
FTT0533c
FTT0555
FTT0557
FTT0558
FTT0878c
FTT0879
FTT1105c
FTT1797c
Dyp-type peroxidase
Porphobilinogen deaminase HemC
Glutaredoxin GrxA
Glutaredoxin
Peroxiredoxin
Short chain dehydrogenase
MsrB
SodC
MsrA1
MsrA
Slide 24
Pattern Mapping of Literature-Based Gene Lists
Iron Acquisition Response Genes
FTT0025c
FTT0026c
FTT0027c
FTT0028c
FTT0029c
FTT0030c
FslE
FslD
FslC
FslB
FslA
fur
Slide 25
Summary
• Array data acquisition finished
• qPCR verification ongoing
• Correlations with literature based gene
selections is variable
Slide 26
Action Items: For Mitch Magee and
Phillip Stafford
• 1.
Provide text describing how to address
the statistical verification of the pooled data sets
• 2.
Prepare some pattern shifting (moving
time points between the two species
experiments) to see if a better correlation can be
achieved as the data are compared. The
differences between the mouse and rat may be
minimized of the patterns are simply timeshifted.
Slide 27