Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profile of T. thermophilus HB8 strain lacking mutS, mutL, or mutS2 gene

ABSTRACT: We analyzed the expression profile of mutS, mutL, or mutS2 deletion mutant strain of Thermus thermophils HB8 during the exponential growth phase. When compared with wild type using t-test (P < 0.01), 8, 111, and 18 genes were over 2-fold up-regulated in mutS, mutL, and mutS2-lacking strains, respectively. Keywords: cell type comparison Wild-type and 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 mutant strains 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 < 0.01 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.

ORGANISM(S): Thermus thermophilus HB8

SUBMITTER: Kenji Fukui

PROVIDER: E-GEOD-22567 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Inactivation of the DNA repair genes mutS, mutL or the anti-recombination gene mutS2 leads to activation of vitamin B1 biosynthesis genes.

Fukui Kenji K Wakamatsu Taisuke T Agari Yoshihiro Y Masui Ryoji R Kuramitsu Seiki S

PloS one 20110428 4

Oxidative stress generates harmful reactive oxygen species (ROS) that attack biomolecules including DNA. In living cells, there are several mechanisms for detoxifying ROS and repairing oxidatively-damaged DNA. In this study, transcriptomic analyses clarified that disruption of DNA repair genes mutS and mutL, or the anti-recombination gene mutS2, in Thermus thermophilus HB8, induces the biosynthesis pathway for vitamin B(1), which can serve as an ROS scavenger. In addition, disruption of mutS, mu ...[more]

PMID: 21552516

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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 compared the expression profile of TTHA1939 deletion mutant strain of Thermus thermophils HB8 with that of wild-type. The mutant strain grown in minimum essential medium (CS medium) exhibited growth arrest and cellular aggregation during the logarithm growth phase. In this phase, 99 genes were suppressed and 63 genes were activated for the mutant strain. The suppressed genes included genes of ribosomal proteins, TCA cycle, amino acid biosynthesis, fatty acid biosynthesis, cobalamin biosynthesis, transporters, and cell division including dnaB and ftsZ. The activated genes included genes of nitrogen metabolism, stress proteins, and proteases. We suggested that these transcriptional alterations in the mutant cell were induced by the ppGpp accumulation which was prevented by ppGpp degradation activity of TTHA1939 in WT cell. Keywords: gene deletion, minimum essential medium, logarithm growth phase Wild-type and the mutant strains were precultured in rich medium (TR medium) for two times at 70 oC and then subcultured to minimum essential medium (CS medium). These cells were harvested during the logarithm growth phase (800 minutes from the start of culture). Total RNA were extracted from each strain and used for the cDNA synthesis. For the biological replication, above experiments were performed four times independently. The cDNA fragments were labeled with biotin-dideoxy UTP, using terminal transferase according to the manufacturerâs instructions (ENZO Biochem. Inc., Farmingdale, NY). The 3â-terminal-labeled cDNA (2 micro g) was hybridized to a TTHB8401 GeneChip (Affymetrix, Santa Clara, CA). The Probe Array was scanned with a GeneArray Scanner (Affymetrix), and then, the image data was scaled to the target intensity by one-step Tukeyâs biweight algorithm using GeneChip Operating software, version 1.0 (Affymetrix, Santa Clara, CA). The data analysis was performed by using GeneSpring GX (Agilent Tech.). The genes which had detection call of âpresenceâ 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 < 0.1 in the Studentâs t-test were extracted. Among them, the genes whose expression level was different more than 2 fold between two strains were considered as significant.

Dataset Information

Transcription profile of T. thermophilus HB8 strain lacking mutS, mutL, or mutS2 gene

Publications

Inactivation of the DNA repair genes mutS, mutL or the anti-recombination gene mutS2 leads to activation of vitamin B1 biosynthesis genes.

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