Unknown,Transcriptomics,Genomics,Proteomics

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MRNA expression under alkylating condition in wild-type strain and ttha1564 deletion mutant of Thermus thermophilus HB8

ABSTRACT: We analyzed the expression profile of wild-type strain and ttha1564 deletion mutant strain of Thermus thermophils HB8 under alkylating condition. In wild type strain, 107 genes and 96 genes were over 2-fold up- and under 0.5-fold down-regulated by alkylation, respectively. In ttha1564 deletion mutant, 35 genes and 12 genes were over 2-fold up- and under 0.5-fold down-regulated by alkylation, respectively. Keywords: stress response comparison The T. thermophilus HB8 wild-type and delta ttha1564 strains were cultured in TT medium (0.4%(w/v) polypeptone, 0.2%(w/v) yeast extract, 0.1%(w/v) NaCl, 0.4 mM CaCl2, and 0.4 mM MgCl2, (pH 7.5)) at 70M-BM-0C until the OD600 reached 0.8. The cultures were then divided into two flasks and an equal volume of TT medium prewarmed at 70M-BM-0C was added. One of the media culture of each strain contained 100 micro g/ml MNNG. We confirmed that the cells can continue to grow in TT medium containing 100 micro g/ml MNNG but DNAs are alkylated sufficiently (Onodera, 2011). After further cultivation at 70M-BM-0C for 10 min, the diluted cultures were collected into an equal of 100% ethanol and stored at -80M-BM-0C. The procedures used for extraction of crude RNA, synthesis of cDNA, labeling of cDNA fragment, hybridization to a TTHB8401a520105F GeneChip (Affymetrix) and data analysis were basically the same as those described previously (Agari et al., 2008; Shinkai et al., 2007). To determine the mRNA expression level of each sample, image data for the three independent samples were processed. stress response comparison

ORGANISM(S): Thermus thermophilus HB8

SUBMITTER: Hiromasa Ohyama

PROVIDER: E-GEOD-27818 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We analyzed the expression profile of mutS 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 70M-BM-0C and then subcultured to 1000 mL of rich medium. These cells were cultured at 70M-BM-0C 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 manufacturerM-bM-^@M-^Ys instructions. The 3M-bM-^@M-^Y-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 TukeyM-bM-^@M-^Ys 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 M-bM-^@M-^\presenceM-bM-^@M-^] 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 StudentM-bM-^@M-^Ys 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.

Project description:We analyzed the expression profile of mutL deletion mutant strain of Thermus thermophils HB8 during the exponential growth phase. Keywords: cell type comparison Wild-type and the mutant strains were precultured in 3 mL of rich medium for two times at 70M-BM-0C and then subcultured to 1000 mL of rich medium. These cells were cultured at70M-BM-0C 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 manufacturerM-bM-^@M-^Ys instructions. The 3M-bM-^@M-^Y-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 TukeyM-bM-^@M-^Ys 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 M-bM-^@M-^\presenceM-bM-^@M-^] 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 StudentM-bM-^@M-^Ys 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.

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MRNA expression under alkylating condition in wild-type strain and ttha1564 deletion mutant of Thermus thermophilus HB8

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