Journal: G3: Genes|Genomes|Genetics

Article Title: Regulation of thiamine and pyruvate decarboxylase genes by Pdc2 in Nakaseomyces glabratus ( Candida glabrata ) is complex

doi: 10.1093/g3journal/jkae132

Figure Lengend Snippet: Fluorescence anisotropy characterizing the protein–DNA interaction between purified Ng Pdc2 DBD and the important cis elements in the NgTHI20 and S cPDC5 promoters. The 55-kDa Ng Pdc2 DBD protein was titrated at different concentrations (µM) with 180 nM of fluorescein-labeled promoter DNA to generate a binding curve and assess the dissociation constant ( K d ) value for the protein–DNA interaction. K d is reported in the figure legend in nanomolars, and the 95% confidence interval is reported in parentheses. As a control, a 38-kDa protein that does not bind DNA was also titrated at different concentrations (µM) with 180 nM of the labeled ScPDC5 promoter DNA. Data shown are the mean and SD of 3 technical replicates.

Article Snippet: To purify the DBD protein using the N-terminal His 10 tag, the Ng Pdc2 DBD-pET16b plasmid was transformed into T7 Express lysY/I q competent Escherichia coli (New England Biolabs).

Techniques: Fluorescence, Purification, Labeling, Binding Assay, Control

Journal: G3: Genes|Genomes|Genetics

Article Title: Regulation of thiamine and pyruvate decarboxylase genes by Pdc2 in Nakaseomyces glabratus ( Candida glabrata ) is complex

doi: 10.1093/g3journal/jkae132

Figure Lengend Snippet: Pdc2 binding in S. cerevisiae and N. glabratus promoter regions. ChIP was performed on strains where ScPDC2 and NgPDC2 were epitope tagged with c-Myc, as well as on untagged wild-type S. cerevisiae and N. glabratus strains, and grown in high and no thiamine growth conditions. qPCR was used to assess the enrichment of Pdc2 at DNA corresponding to the THI promoters in S. cerevisiae and N. glabratus as well as a gene not regulated by thiamine ( ACT1 ) as a negative control. Data are presented as the ratio of immunoprecipitated DNA from Pdc2-Myc-tagged strains relative to the untagged wild-type strain for each species. Data shown are the average and SE from 3 independent ChIP experiments performed for all strains. A 1-way ANOVA with a post hoc Tukey's multiple comparisons test was performed.

Article Snippet: To purify the DBD protein using the N-terminal His 10 tag, the Ng Pdc2 DBD-pET16b plasmid was transformed into T7 Express lysY/I q competent Escherichia coli (New England Biolabs).

Techniques: Binding Assay, Negative Control, Immunoprecipitation

Journal: G3: Genes|Genomes|Genetics

Article Title: Regulation of thiamine and pyruvate decarboxylase genes by Pdc2 in Nakaseomyces glabratus ( Candida glabrata ) is complex

doi: 10.1093/g3journal/jkae132

Figure Lengend Snippet: Ng Pdc2 is binding to the promoter regions that contain the proposed cis elements for NgTHI20 , NgTHI10 , and NgPET18 , and Ng Thi3 appears to colocalize at these promoters with Pdc2. ChIP was performed on strains where NgPDC2 and NgTHI3 were epitope tagged with c-Myc, as well as on an untagged N. glabratus wild-type strain and grown in no thiamine growth conditions. Enrichment of N. glabratus Pdc2 and Thi3 at promoters was quantified using next-generation sequencing. Coverage for various THI and PDC promoters was plotted against the nucleotide position 1,000-bp upstream and downstream of the ATG, with 6 replicates of N. glabratus wild type and Ng Pdc2-Myc and 3 replicates of Ng Thi3-Myc. There is a peak in coverage for Ng Pdc2-Myc and Ng Thi3-Myc corresponding to the conserved cis sequences found to be required for upregulation of THI20 , THI10 , and PET18 (indicated by a line and asterisk), while the untagged wild-type strain shows no peak. There are no peaks for Ng Pdc2-Myc or Ng Thi3-Myc corresponding to the known cis element important for regulation of the NgPMU3 promoter (indicated by a line and asterisk).

Article Snippet: To purify the DBD protein using the N-terminal His 10 tag, the Ng Pdc2 DBD-pET16b plasmid was transformed into T7 Express lysY/I q competent Escherichia coli (New England Biolabs).

Techniques: Binding Assay, Next-Generation Sequencing