Journal:

Article Title: Development and Evaluation of Functional Gene Arrays for Detection of Selected Genes in the Environment

doi: 10.1128/AEM.67.12.5780-5790.2001

Figure Lengend Snippet: Quantitative analysis of functional gene arrays. (A) Relationship of hybridization signal intensity to DNA target concentration from a single pure culture. Genomic DNA from nirS-containing P. stutzeri E4-2 was labeled with Cy5 and hybridized to the microarrays at the following target concentrations: 0.5, 1, 2.5, 5, 10, 25, 50, and 100 ng. The plot shows the log-transformed average hybridization intensity versus the log-transformed target DNA concentration. (B) Relationship of hybridization signal intensity to DNA target concentration using a mixture of target DNAs. The PCR products from the following nine strains were mixed together in different quantities (in picograms): E4-2 (nirS), 1,000; G179 (nirK), 500; wc301–37 (amoA), 250; ps-47 (amoA), 125; pB49 (nirS), 62.5; Y32K (nirK), 31.3; wA15 (nirS), 15.6; ps-80 (amoA), 7.8; wB54 (nirK), 3.9. All of these genes are less than 80% identical. The mixed templates were labeled with Cy5. The plot shows the log-transformed average hybridization intensity versus the log-transformed target DNA concentration for each strain. (C) Effects of probe DNA size and composition on hybridization signal intensity. Microarrays contained DNA fragments (200 ng μl−1) of different sizes amplified from different regions in the S. oneidensis MR-1 genome. The target DNA was prepared by labeling MR-1 genomic DNA with Cy5 using the Klenow fragment with random hexamer primers. For panels A and B, the data points are mean values derived from three independent microarray slides, with three replicates on each slide (a total of nine data points). Error bars showing the standard deviations are presented.

Article Snippet: Yeast genomic DNA was prepared from Saccharomyces cerevisiae ATCC 18824.

Techniques: Functional Assay, Hybridization, Concentration Assay, Labeling, Transformation Assay, Amplification, Random Hexamer, Derivative Assay, Microarray

Journal:

Article Title: Development and Evaluation of Functional Gene Arrays for Detection of Selected Genes in the Environment

doi: 10.1128/AEM.67.12.5780-5790.2001

Figure Lengend Snippet: Array hybridization images showing the detection sensitivity with labeled pure genomic DNA and bulk community DNA from soil. (A) Genomic DNA from a pure culture of nirS-containing P. stutzeri E4-2 was labeled with Cy5 using the random primer labeling method. The target DNA was hybridized to the microarrays at total concentrations of 0.5, 1, and 5 ng. (B) Genomic DNA from surface soil O22 (10, 25, and 50 ng) was labeled with Cy5 as described for panel A and hybridized with the microarrays.

Article Snippet: Yeast genomic DNA was prepared from Saccharomyces cerevisiae ATCC 18824.

Techniques: Hybridization, Labeling

Journal: Nucleic Acids Research

Article Title: H2B ubiquitylation and the histone chaperone Asf1 cooperatively mediate the formation and maintenance of heterochromatin silencing

doi: 10.1093/nar/gkx422

Figure Lengend Snippet: Cells lacking both anti-silencing function 1 (Asf1) and H2Bub exhibit defects in stress-resistance and gene silencing. ( A ) Specific genetic interactions between H2Bub and the Asf1 histone chaperone. Cell growth was observed for 10-fold serial dilutions of yeast cells spotted onto non-selective YPD plates, or plates containing one of the following chemicals at 30°C for 3–5 days: methyl methanesulfonate (MMS; causes DNA damage), hydroxyurea (HU; source of replication stress), UV irradiation (leads to DNA damage) or 6-Azauracil (6-AU; affects transcriptional elongation). The rad52Δ strain served as a control for MMS, HU and UV treatment, while rtf1Δ served as a control for 6-AU treatment. ( B ) Positional cluster analysis shows gene expression changes in asf1Δ, htb-K123R and asf1Δ htb-K123R mutant cells. Only genes whose expression was increased or decreased by at least 1.75-fold in at least one of these strains are shown (775 upregulated genes and 826 downregulated genes). Genes located within 20 kb of their respective telomeres are marked on the left as the telomere region. ( C ) H2Bub and Asf1 are required for gene silencing within the proximity of telomeres. Transcripts from each strain were isolated and analyzed by Phalanx Yeast OneArray ®. The numbers of genes affected in the mutants compared to wild-type (WT) were plotted against their position from telomeres in kilobase pairs (kb). The numbers of upregulated and downregulated genes are shown as red and green bars, respectively.

Article Snippet: DNA microarray analysis was performed with Phalanx Yeast OneArray ® chip (Phalanx Biotech).

Techniques: Irradiation, Control, Gene Expression, Mutagenesis, Expressing, Isolation

Journal: Nucleic Acids Research

Article Title: H2B ubiquitylation and the histone chaperone Asf1 cooperatively mediate the formation and maintenance of heterochromatin silencing

doi: 10.1093/nar/gkx422

Figure Lengend Snippet: H2Bub and Asf1 are important for cellular response to sex pheromone. ( A ) Loss of mating type silencing in asf1Δ htb-K123R mutant cells (Halo assay). The indicated strains (mating type a) were treated with various doses of α-factor (1, 5 and 25 mg/ml) and the inhibition of cell growth indicates that mating type silencing was well maintained. ( B ) Cells lacking both Asf1 and H2Bub cannot respond to alpha factor. Isogenic WT (HTB1), htb-K123R, asf1Δ and asf1Δ htb-K123R cells were arrested at G1 phase with α-factor at 30°C for 2–5 h; exp: cells at exponential stage; h: the time in hours under α-factor treatment. Cells at each time point were stained with SYBR Green and the DNA content was determined by flow cytometry. ( C ) Transcriptional profiles of htb-K123R, asf1Δ and asf1Δ htb-K123R mutants under α-factor treatment. Cells were treated with α-factor for 3 h and transcripts were isolated and analyzed by Phalanx Yeast OneArray ® . Red and green represent upregulated and downregulated genes, respectively. Genes that were not significantly affected appear black in the heat map. Genes that exhibited a 1.75-fold change or greater between the WT and at least one of the mutants were selected and used in gene clustering analyzes. Table A: downregulated genes were clustered into two predominant GO terms, sexual reproduction and response to pheromone . Table B: upregulated genes were clustered into several GO terms.

Article Snippet: DNA microarray analysis was performed with Phalanx Yeast OneArray ® chip (Phalanx Biotech).

Techniques: Mutagenesis, Halo Assay, Inhibition, Staining, SYBR Green Assay, Flow Cytometry, Isolation

Journal: Nature communications

Article Title: Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance

doi: 10.1038/ncomms4446

Figure Lengend Snippet: (a) Sod1 is required for the induction of oxidative response (OR) genes. WT or sod1 Δ cells were treated without or with 0.4 mM H 2 O 2 for 20 min and analyzed for global gene expression profile. 123 Sod1-dependent genes were identified and most of the known genes belong to five related functional categories. (b) Shown is the relative induction level of OR genes by H 2 O 2 in each category in WT and sod1 Δ cells. Data represents average fold change of induction in each category. (c) Shown is the heat map of genes in the oxidative stress response category. (d) Validation of representative genes ( GRE2 , Genes de Respuesta a Estres 2; TSA2 : Thiol-Specific Antioxidant 2; YML131; STF2 : STabilizing Factor 2) in the oxidative stress response category by RT-qPCR. Error bars indicate ± SD from triplicates of two independent experiments. * p < 0.05. (e) Nuclear Sod1 is critical for the induction of OR genes. Yeast cells expressing different forms of Sod1 were treated with 0.4 mM H 2 O 2 for 20 min. Representative genes were validated by RT-qPCR. Error bars indicate ± SD from triplicates of two independent experiments. * p < 0.05. (f) The induction of OR genes by ROS was attenuated in Sod1 S60,99A cells. Yeast cells expressing Sod1 or Sod1 S60,99A were treated with 0.4 mM H 2 O 2 for 20 min. Expression of GRE2 and RNR3 were determined by RT-qPCR. Error bars indicate ± SD from triplicates of two independent experiments. * p < 0.05. (g) ROS treatment increases the association of Sod1 with promoter of oxidative responsive genes. WT (SZy1051) and sod1 Δ (SZy1050) cells were treated with 0.4 mM H 2 O 2 for 20 min. The binding of Sod1 to representative promoters were analyzed by chromatin immunoprecipitation (ChIP). (h) Quantification of the Fig. 5g experiment. Error bars indicate ± SD from triplicates of two independent experiments. * p < 0.05, Student’s t -test.

Article Snippet: DNA microarray analysis of yeast global gene expression was carried out by Rutgers RUCDR Analytical and Informatics Services using the following procedure.

Techniques: Gene Expression, Functional Assay, Biomarker Discovery, Quantitative RT-PCR, Expressing, Binding Assay, Chromatin Immunoprecipitation