Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Comparative expression analysis between PfmR (TTHB023) deletion mutant and wild-type of Thermus thermophilus HB8

ABSTRACT: We observed the expression profile of the total mRNA of PfmR (TTHB023) deletion mutant of Thermus thermophilus HB8 strain grown in a rich (TT) medium at 70M-BM-0C compared with that of wild type. Seven wild-type and three mutant T. thermophilus HB8 strains were each pre-cultured at 70M-BM-0C for 16 h in 4 ml of TT medium containing 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, 0.4 mM CaCl2, and 0.4 mM MgCl2, which was adjusted to pH 7.2 with NaOH. The cells were inoculated into 1 liter of the same medium and then cultivated at 70M-BM-0C. Cells were collected after 360 min, and then crude RNA was extracted. In order to compare mRNA expression in the PfmR deletion mutant with that of wild type, the expression of each mRNA was analyzed on a GeneChip.

ORGANISM(S): Thermus thermophilus HB8

SUBMITTER: Akeo Shinkai

PROVIDER: E-GEOD-36909 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Transcriptional repression mediated by a TetR family protein, PfmR, from Thermus thermophilus HB8.

Agari Yoshihiro Y Sakamoto Keiko K Kuramitsu Seiki S Shinkai Akeo A

Journal of bacteriology 20120629 17

PfmR is one of four TetR family transcriptional regulators found in the extremely thermophilic bacterium, Thermus thermophilus HB8. We identified three promoters with strong negative regulation by PfmR, both in vivo and in vitro. PfmR binds pseudopalindromic sequences, with the consensus sequence of 5'-TACCGACCGNTNGGTN-3' surrounding the promoters. According to the amino acid sequence and three-dimensional structure analyses of the PfmR-regulated gene products, they are predicted to be involved ...[more]

PMID: 22753056

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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.

Dataset Information

Comparative expression analysis between PfmR (TTHB023) deletion mutant and wild-type of Thermus thermophilus HB8

Publications

Transcriptional repression mediated by a TetR family protein, PfmR, from Thermus thermophilus HB8.

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