DIAMONDS midterm review

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• DIAMONDS midterm review
• Workpackage 1
• Dept. Plant Systems Biology
• VIB/Univ. Ghent
• Brussels, 27 September 2006

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Cell cycle transcript profiling

• thaliana cell culture, aphidicolin block, 10 time
  points
• Synchronization quality checked by qPCR (30
  genes)
• 2 biological repeats, 4 technical repeats,
  interwoven loop design
• CATMA arrays
• Unsatisfactory hybridization quality

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Genetic perturbations

• Focus on the transition to endocycle
• Transgenic lines
• DEL1 knockout
• DEL1 over-expression (2 lines)
• CCS52A1 knockout
• CDKB11 dominant negative mutant (2 lines)
• KRP2 over-expression (2 lines)
• Leaves of 15 days old plantlets
• Affimetrix ATH1 chips (24 in total)
• Preliminary results the expression of 50 genes
  was consistently correlated with the extent of
  endocycling across all the 5 perturbations

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DNA damage checkpoint

• Substituted with DNA replication checkpoint
• thaliana root tips, WT Col-0 and Wee1 KO
• Hydroxyurea treatment
• 3 time-points 0h, 5h, 24h
• 4 biological repeats, pooled pair wise
• Affimetrix ATH1 arrays, 24 in total

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DNA replication checkpoint
Preliminary results (24h time point only)
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Cell cycle exit/entry in response to nutrients
availability
Phosphate and nitrogen starvation experiments
performed elsewhere No experiments planned
Compensated with genetic perturbation
experiments
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Cell cycle exit/entry in response to
extracellular signaling
Response to auxin in the pericycle
3 days old Arabidopsis seedlings, WT and slr1
mutant FACS separated pericycle cells 2 time
points 2h and 6h 2 biological repeats
Affimetrix ATH1 arrays, 14 in total 1123
differentially expressed genes
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Cell cycle exit/entry in response to
extracellular signaling
Response to jasmonate
Arabidopsis suspension culture 3 time points
0.5h, 2h and 6h 2 biological repeats
Affimetrix ATH1 arrays 598 genes
differentially expressed at one or more time
points In 1 of the 8 clusters cell cycle genes
are over-represented
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Proteome analysis
TAP Technology fully established, 3
baits/week Goal 125 baits 14 baits fully
processed, 2 baits no interactions, 12 baits 2-50
interactions 65 baits in the pipeline, 50 more
will be chosen among the preys Y2H Goal - a
matrix of interactions for 80 cell cycle
proteins Pray and bait constructs for 60
proteins
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Functional validation and characterization
Putative targets of E2F/DP transcription factors
181 putative E2F target genes (Plant Physiol.
2005 Sep139(1)316-28) T-DNA insertions were
identified in 22 putative targets genes
Mutations are being verified by expression
analysis
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Functional validation and characterization
Proliferation associated genes
131 genes expressed specifically in proliferating
Arabidopsis cells (Plant Physiol. 2005
Jun138(2)734-43 ) Analysis focused on genes of
unknown function 15 genes selected 3 small
families 7 unique genes (all possessing
mammalian homologues) T-DNA insertions found in
14 genes (42 lines screened), KO verified for 5
genes 3 display growth phenotypes Complementary
approach amiRNA, constructs are ready
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