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. 2000 Mar 14;97(6):2456–2461. doi: 10.1073/pnas.97.6.2456

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Copyright © 2000, The National Academy of Sciences

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The mVDAC3 and Gadd45 promoters display temperature-sensitive transcription in ts13 cells. (A) The promoter region of the mVDAC3 gene is shown, along with the location of putative Sp1 binding sites (shaded boxes) (21). Primer extension analysis was performed with total RNA purified from BHK-21 cells (lane 5) and ts13 (lane 6) cells after incubation of synchronized growing cells at the restrictive temperature for 24 hours. The corresponding DNA sequence reactions were analyzed on the same gel and are shown in lanes 1–4. The sequence surrounding the −30 region relative to the transcription start site is shown. (B) A construct containing the mVDAC3 promoter (−316 to +301) fused upstream of the CAT reporter was transiently transfected into BHK-21 or ts13 cells with an SV40-Renilla construct whose activity served as an internal standard and, where indicated, with a construct that expresses wild-type hTAF_II250. Cells were incubated at the indicated temperature for 24–30 hours and were harvested, and CAT and Renilla activity was assayed. The activity of this reporter in BHK-21 cells at 33°C was standardized to 1.0, and all other values, along with their associated standard deviation, are presented relative to this. (C) Deletion mapping of the mVDAC3 promoter to identify TAF_II250 responsive elements. The indicated deletions were fused upstream of the CAT reporter gene and were transiently transfected into BHK-21 and ts13 cells as described in B. The relative temperature sensitivity of each promoter (with its associated standard deviation), which is presented as a ratio of the level of activity in ts13 cells to the level of activity in BHK-21 cells, is shown. A lower number represents a greater reduction of expression in ts13 cells. The relative activity of each promoter in BHK-21 cells is also shown. (D) Transcriptional activity of hybrid mVDAC3/Gadd45 promoters in ts13 and BHK-21 cells. Constructs contained either the Gadd45 core promoter (−40 to +60), the mVDAC3 upstream enhancer region (−120 to −40) fused to the Gadd45 core promoter (−40 to +60), or the Gadd45 upstream enhancer region (−849 to −40) fused upstream of the mVDAC3 core promoter (−40 to +60). All promoters were inserted directly upstream of the CAT reporter. These constructs were transiently transfected into BHK-21 and ts13 cells, as described in B. The relative temperature sensitivity of each promoter is shown, which is presented as a ratio of the level of activity in ts13 cells relative to the level in BHK-21 cells. A number less than 1.0 represents a greater reduction of expression in ts13 cells relative to BHK-21 cells whereas a number greater than 1.0 represents a greater activity in ts13 cells relative to BHK-21 cells. The relative activity of the Gadd45 promoters is also shown. NA, not applicable because these are promoter fusions.

The mVDAC3 and Gadd45 promoters display temperature-sensitive transcription in ts13 cells. (A) The promoter region of the mVDAC3 gene is shown, along with the location of putative Sp1 binding sites (shaded boxes) (21). Primer extension analysis was performed with total RNA purified from BHK-21 cells (lane 5) and ts13 (lane 6) cells after incubation of synchronized growing cells at the restrictive temperature for 24 hours. The corresponding DNA sequence reactions were analyzed on the same gel and are shown in lanes 1–4. The sequence surrounding the −30 region relative to the transcription start site is shown. (B) A construct containing the mVDAC3 promoter (−316 to +301) fused upstream of the CAT reporter was transiently transfected into BHK-21 or ts13 cells with an SV40-Renilla construct whose activity served as an internal standard and, where indicated, with a construct that expresses wild-type hTAF_II250. Cells were incubated at the indicated temperature for 24–30 hours and were harvested, and CAT and Renilla activity was assayed. The activity of this reporter in BHK-21 cells at 33°C was standardized to 1.0, and all other values, along with their associated standard deviation, are presented relative to this. (C) Deletion mapping of the mVDAC3 promoter to identify TAF_II250 responsive elements. The indicated deletions were fused upstream of the CAT reporter gene and were transiently transfected into BHK-21 and ts13 cells as described in B. The relative temperature sensitivity of each promoter (with its associated standard deviation), which is presented as a ratio of the level of activity in ts13 cells to the level of activity in BHK-21 cells, is shown. A lower number represents a greater reduction of expression in ts13 cells. The relative activity of each promoter in BHK-21 cells is also shown. (D) Transcriptional activity of hybrid mVDAC3/Gadd45 promoters in ts13 and BHK-21 cells. Constructs contained either the Gadd45 core promoter (−40 to +60), the mVDAC3 upstream enhancer region (−120 to −40) fused to the Gadd45 core promoter (−40 to +60), or the Gadd45 upstream enhancer region (−849 to −40) fused upstream of the mVDAC3 core promoter (−40 to +60). All promoters were inserted directly upstream of the CAT reporter. These constructs were transiently transfected into BHK-21 and ts13 cells, as described in B. The relative temperature sensitivity of each promoter is shown, which is presented as a ratio of the level of activity in ts13 cells relative to the level in BHK-21 cells. A number less than 1.0 represents a greater reduction of expression in ts13 cells relative to BHK-21 cells whereas a number greater than 1.0 represents a greater activity in ts13 cells relative to BHK-21 cells. The relative activity of the Gadd45 promoters is also shown. NA, not applicable because these are promoter fusions.