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| Status |
Public on Sep 04, 2006 |
| Title |
Time course response of Synechocystis PCC 6803 to UV irradiation |
| Organism |
Synechocystis sp. PCC 6803 |
| Experiment type |
Expression profiling by array
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| Summary |
The responses of the transcriptome of Synechocystis PCC 6803 to UV-irradiation were measured at time points over 36 h. Irradiation was provided by Sylvania soft white DuluzR compact fluorescent 23W bulbs (Osram Sylvania Ltd, Mississauga, Canada), a 20W RS UV-B medical light with a spectral maximum at 310 nm (model ‘TL’, Philips, Holland), and 15W black lights each with a spectral maximum at 368 nm (model F15T8-BL, General Electric, USA). Total quantum scalar irradiance was measured with a model QSL-100 meter (Biospherical Instruments Inc., San Diego, CA). The flux densities of the UV-A and UV-B components of the spectrum were measured with DIX series UV-B and 365A sensors, respectively, with a Spectroline DRC-100X digital radiometer (Spectronics Corporation, Westbury, NY). In these experiments full illumination represented a continuous photon flux density in the visible range of 330 μmol photons m-2 s-1, with UV-A and UV-B maxima of 3.8 x 10^6 and 0.8 x 10^6 mW m-2, respectively. All values reported were the incident fluxes within culture vessels at the immediate surface of the cell suspensions. Aliquot cultures (in duplicate) were harvested after 0, 15 min, 1 h, 3 h, 6 h, 12 h, 24 h and 36 h of UV-irradiation. For each time point, total RNA were isolated from stressed and unstressed cells, reverse-transcribed, differentially labelled (dye swapped), hybridized together (stressed versus unstressed samples) and analyzed with DNA glass microarrays (two slides per each time point) (Custom-commercial array : CyanoCHIP version 2.0, TAKARA). To identify differentially expressed genes, the median of the normalized ratio of Cy5/Cy3 intensity was calculated for each spot of the replicated dye-swap. The results of the analysis were carefully examined to exclude the dye effect between the 2 Cy-swapped arrays.
Keywords: UV-irradiation, desiccation, Synechocystis PCC 6803, cyanobacteria, time course, transcription
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| Overall design |
Goal -
Compare the responses of cyanobacterium Synechocystis sp. PCC 6803 to UV-irradiation and dehydration/desiccation stress.
Origin of biological sample -
The axenic strain Synechocystis sp. PCC 6803 was obtained from the American Type Culture Collection (ATCC 27184). Special attention was given to maintain all the Synechocystis cultures under the same conditions to minimize environmental variation.
Brief description -
Ultraviolet (UV) light and desiccation stresses often co-occur in natural environments, making their combined effects important selective factors within microbial populations. The effects of these stresses on the cyanobacterium Synechocystis sp. PCC 6803 was assessed through transcriptional analyses using differential display and microarray assays.
Experimental factors and design-
Axenic Synechocystis cultures were grown at 25°C in BG-11 medium, on a rotary shaker at 70 rpm, at a photon flux density of 200 µmol photons m-2 s-1. At the midpoint of the exponential phase of growth (~ 10^6 to 10^7 cells ml-1), 25-ml aliquots of the cell suspension were subjected to UV-irradiation (in 50-ml flasks) with a continuous photon flux density in the visible range of 330 µmol photons m-2 s-1. UV-A and UV-B maxima were 3.8 x 10^6 and 0.8 x 10^6 mW m-2, respectively. Cells were harvested after 0, 15 min, 1 h, 3 h, 6 h, 12 h, 24 h and 36 h of irradiation for disruption and rapid recovery of RNA. As controls, RNA was obtained from Synechocystis cells grown without UV-irradiation. Cells were disrupted with glass beads and RNA was extracted with a buffer of acidified phenol (65°C), sodium acetate pH 5.1, EDTA and sodium dodecyl sulfate), followed by ethanol precipitation. The RNA (10 µg) was annealed with 20 µg Random RT primer and reverse transcription into cDNA was achieved with Superscript II reverse transcriptase enzyme for 2h, at 42°C. The assay contained dGTP, dCTP, dATP and dTTP, each a final concentration of 0.5 mM. After hydrolysis of RNA with 0.1M NaOH and 10 mM EDTA, then neutralization, cDNAs were purified using a Quiagen QIAquick PCR purification kit as modified in the Genisphere 3DNA Array 350RP protocol.
Two experimental replicates of RNAs from time points 12 ml, and two experimental replicates from 0.5 ml, of dehydration, were used for hybridization to microarrays. Three experimental replicates of RNAs from time point 3h of UV-irradiation, and three experimental replicates from time point 36h of UV-irradiation, were used for hybridization to microarrays.
Quality contro-l
DNAse treatment (DNAse free, Ambion) was for 3 h at 37 °C. This treatment was sometimes repeated for 2 h until the absence of any detectable DNA was checked by the absence of product after 40 cycles of PCR amplification. The quality and purity of RNAs was judged by A254/280 (1.9 to 2.1) and by the integrity of 23S and 16S rRNA resolved through gel electrophoresis. The ribosomal subunits gave sharp and bright bands that were comparable among all samples. The quality of the random primed cDNA samples was checked by SDS-PAGE after ligation of Cy3/Cy5 cap sequences. Test arrays were performed to determine the optimum hybridization and washing conditions of the TAKARA’s microarray chips.
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| Contributor(s) |
Garczarek L, Ramakrishan N, Kumar D, Helm RF, Potts M |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Apr 04, 2006 |
| Last update date |
Aug 08, 2013 |
| Contact name |
Malcolm Potts |
| Phone |
540 231-5745
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| Fax |
540 231-9070
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| URL |
http://vigen.biochem.vt.edu
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| Organization name |
Virginia Tech
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| Department |
Biochemistry
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| Lab |
Virginia Tech Center for Genomics
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| Street address |
W. Campus Drive
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| City |
Blacksburg |
| State/province |
VA |
| ZIP/Postal code |
24061 |
| Country |
USA |
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| Platforms (1) |
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| Samples (6)
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| GSM102953 |
Synechocystis cells grown under UV-irradiation for 3 hrs. Replicate 1 |
| GSM102954 |
Synechocystis cells grown under UV-irradiation for 3 hrs. Replicate 2 |
| GSM102955 |
Synechocystis cells grown under UV-irradiation for 3 hrs. Replicate 3 |
| GSM102956 |
Synechocystis cells grown under UV-irradiation for 36 hrs. Replicate 1 |
| GSM102957 |
Synechocystis cells grown under UV-irradiation for 36 hrs. Replicate 2 |
| GSM102958 |
Synechocystis cells grown under UV-irradiation for 36 hrs. Replicate 3 |
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| This SubSeries is part of SuperSeries: |
| GSE4613 |
Time course response of Synechocystis PCC 6803 to dehydration/desiccation and UV irradiation |
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| Relations |
| BioProject |
PRJNA104693 |
| Supplementary data files not provided |
| Processed data included within Sample table |
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