plantlet - Seeds were sown on MS255 (Duchefa) + agar (8g/L). they were then put 48h at 4degreeC then placed at 22degreeC Hydrometry : 70% light : 2000 lux, Day/night : 16h/8h.
Extracted molecule
total RNA
Extraction protocol
Pool of extract, CRB.4A:1ug, CRB.2A:1ug, CRB.1A:1ug.
Label
Cy5
Label protocol
labelling Cy3 and Cy5 indirect, amplification=yes, DNA 5 ug. Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).
plantlet - Seeds were sown on MS255 (Duchefa) + agar (8g/L). they were then put 48h at 4degreeC then placed at 22degreeC Hydrometry : 70% light : 2000 lux, Day/night : 16h/8h.
Extracted molecule
total RNA
Extraction protocol
Pool of extract, WS.2A:1ug, WS.1A:1ug, WS.4A:1ug.
Label
Cy3
Label protocol
labelling Cy3 and Cy5 indirect, amplification=yes, DNA 5 ug. Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).
Hybridization protocol
CRB Cy5 / WS Cy3 : 40pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.
Scan protocol
GenePix Pro 3.0, Cy3:pmt voltage 532nm,700V,laser power 100%, Cy5:635nm,pmt voltage 650V,laser power 100%
Description
Cullin proteins belong to a multigene family that includes at least three members in budding yeast, six in human, five in C. elegans and five in Arabidopsis thaliana. All cullins analyzed so far directly interact with RBX1/HRT1/ROC1, a RING finger protein, thereby forming the core module of different ubiquitin ligase complexes, which specifically recruit substrate proteins for ubiquitylation and subsequent degradation by the proteasome. CULLIN1, the only cullin protein studied so far in plants, has been shown to be essential for plant development and to play a role in multiple signalling cascades. Here, we report on the first molecular and genetic characterisation of a plant Cullin 3. In contrast to fungi and animals, the genome of the model plant Arabidopsis thaliana contains two related CULLIN 3 genes, called CUL3A and CUL3B. We found that CUL3A is ubiquitously expressed in plants and that the CUL3A protein is also able to interact with RBX1 and certain Arabidopsis BTB-domain proteins. In order to determine the role of CUL3A in plant development, we used a reverse genetic approach and identified T-DNA insertion mutants. cul3a null mutants flower slightly latter than the control plants and their hypocotyls exhibit a reduced sensitivity to far red light. The viability of the cul3a mutant plants suggests functional redundancy between both CUL3 genes in plants.
Data processing
The raw data comprised the logarithm of median feature pixel intensity at wavelength 635 nm (red) and 532 nm (green). No background was subtracted. An array-by-array normalization was performed to remove systematic biases. First, we excluded spots that were considered badly formed features by the experimenter (Flags=-100). Then we performed a global intensity-dependent normalization using the loess procedure (see Yang et al., 2002) to correct the dye bias. Finally, on each block, the log-ratio median is subtracted from each value of the log-ratio of the block to correct a print-tip effect on each metablock.
