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E-GEOD-22349 - mRNA expression in mutS2 deletion mutant of Thermus thermophilus HB8
Released on 2 June 2011, last updated on 27 January 2013
Thermus thermophilus HB8
We analyzed the expression profile of mutS2 deletion mutant strain of Thermus thermophils HB8 during the exponential growth phase. Keywords: cell type comparison The mutant strains were precultured in 3 mL of rich medium for two times at 70°C and then subcultured to 1000 mL of rich medium. These cells were cultured at70°C for 300 min , and then cells were harvested from 50 ml of the culture. Total RNA were extracted from wild-type and mutS-lacking strain and used for the cDNA synthesis by SuperScript II (Invitrogen, Carlsbad, CA). The cDNA fragments were labeled with GeneChip DNA Labeling Reagent (Affymetrix, Santa Clara, CA), using terminal transferase according to the manufacturer’s instructions. The 3’-terminal-labeled cDNA was hybridized with a TTHB8401a520105F GeneChip (Affymetrix) which contained probe sets of 25-mer oligonucleotides for 2238 ORFs and 1096 intergenic regions. After washing and staining with streptavidin-phycoerythrin (Invitrogen) by GeneChip Fluidics Station 450XP (Affymetrix), the array was scanned by a GeneChip Scanner 3000 (Affymetrix). The image data was scaled to the target intensity by one-step Tukey’s biweight algorithm using GeneChip Operating software, version 1.2 (Affymetrix). The data analysis was performed on the Subio platform version 1.6 (Subio Inc., Tokyo, Japan). The genes which had detection call of “presence” more than 4 times from 8 samples were used for following analysis. The normalized intensities for the mutant strains were calculated by using the intensities for wild-type strains of each data set, and then, the average of these intensities were used. The genes which produced P value < 0.05 in the Student’s t-test were extracted. Among them, the genes whose expression level was different more than 2 fold between two strains were considered as significant. For the biological replication, above experiments were performed three times independently.
transcription profiling by array
Kenji Fukui <email@example.com>, K Fukui, S Kuramitsu, Y Agari
Inactivation of the DNA repair genes mutS, mutL or the anti-recombination gene mutS2 leads to activation of vitamin B1 biosynthesis genes. Fukui K, Wakamatsu T, Agari Y, Masui R, Kuramitsu S. , PMID:21552516