Fig. 2.
Fig. 2. Deletions made in the 5′ end upstream of initiation start ATG in the human RPS19 gene and measured promoter activities. / (A) We identified 4 consensus regions (▪) between the mouse and human 5′ sequence upstream of the initiation start ATG. The human genomic sequence upstream of the translational initiation start site is represented as the black line at the top of the panel. The predicted promoter found in the human sequence between the second and third consensus regions is also indicated. The consensus binding motifs for putative transcription factors in the consensus regions 1, 3, and 4 are noted with arrowheads. We cloned the full-length human 5′ end (wild-type RPS19 gene at the top of the panel and mutants below) in a promoterless pGL3 vector, which contained a luciferase reporter gene. The 2 mutations found in patients affected by DBA in the 5′ sequence upstream of the translational initiation start site were also cloned into pGL3 vector. Their positions are indicated. We deleted each of the consensus regions, the predicted promoter, both in a same construct the predicted promoter and the consensus region 3, and finally the putative κB element in the consensus region 3. The number of the flanking nucleotide indicates the location of each construct. (B) We measured the activity of the luciferase reporter gene (y-axis) driven by RPS19 5′ end wild-type (WT) and mutated constructs: deletion of the human predicted promoter (Pre Pro), deletion of the consensus region 3 (Del 3), deletion of both consensus region 3 and predicted promoter (Pre Pro+Del 3), deletion of binding motif for potential transcription factors from the NF-κB/Rel family (Del κB element). When we deleted the RPS19 human gene predicted promoter (Pre Pro, 3 clones each tested in 3 independent experiments), we observed a 40% decrease in luciferase reporter gene activity compared with wild-type RPS19 gene. After deletion of the consensus region 3 (Del 3, 3 clones each tested in 3 independent experiments), we obtained a greater decrease in luciferase reporter gene activity (decrease of 66%). More importantly, when we deleted both the predicted promoter and consensus region 3 (Pre Pro+Del 3, 2 clones in 2 independent experiments), we noted an even larger decrease in the luciferase reporter gene (decrease of 86%). The positions of Pre Pro and Del 3 deletion in the 5′ flanking human region upstream of initiation start are indicated at the bottom of the panel. To investigate transcription factors, which may regulate theRPS19 gene promoter activity in the third consensus region, we measured the luciferase reporter gene (y-axis) driven by humanRPS19 in which we deleted the binding motif for potential transcription factors c-Rel/Rel-A (Del κB element, clones 1 and 2). Unexpectedly, the luciferase reporter gene activity was not decreased but in fact increased 3-fold compared with the wild-typeRPS19 gene. Data from individual experiments are shown.

Deletions made in the 5′ end upstream of initiation start ATG in the human RPS19 gene and measured promoter activities.

(A) We identified 4 consensus regions (▪) between the mouse and human 5′ sequence upstream of the initiation start ATG. The human genomic sequence upstream of the translational initiation start site is represented as the black line at the top of the panel. The predicted promoter found in the human sequence between the second and third consensus regions is also indicated. The consensus binding motifs for putative transcription factors in the consensus regions 1, 3, and 4 are noted with arrowheads. We cloned the full-length human 5′ end (wild-type RPS19 gene at the top of the panel and mutants below) in a promoterless pGL3 vector, which contained a luciferase reporter gene. The 2 mutations found in patients affected by DBA in the 5′ sequence upstream of the translational initiation start site were also cloned into pGL3 vector. Their positions are indicated. We deleted each of the consensus regions, the predicted promoter, both in a same construct the predicted promoter and the consensus region 3, and finally the putative κB element in the consensus region 3. The number of the flanking nucleotide indicates the location of each construct. (B) We measured the activity of the luciferase reporter gene (y-axis) driven by RPS19 5′ end wild-type (WT) and mutated constructs: deletion of the human predicted promoter (Pre Pro), deletion of the consensus region 3 (Del 3), deletion of both consensus region 3 and predicted promoter (Pre Pro+Del 3), deletion of binding motif for potential transcription factors from the NF-κB/Rel family (Del κB element). When we deleted the RPS19 human gene predicted promoter (Pre Pro, 3 clones each tested in 3 independent experiments), we observed a 40% decrease in luciferase reporter gene activity compared with wild-type RPS19 gene. After deletion of the consensus region 3 (Del 3, 3 clones each tested in 3 independent experiments), we obtained a greater decrease in luciferase reporter gene activity (decrease of 66%). More importantly, when we deleted both the predicted promoter and consensus region 3 (Pre Pro+Del 3, 2 clones in 2 independent experiments), we noted an even larger decrease in the luciferase reporter gene (decrease of 86%). The positions of Pre Pro and Del 3 deletion in the 5′ flanking human region upstream of initiation start are indicated at the bottom of the panel. To investigate transcription factors, which may regulate theRPS19 gene promoter activity in the third consensus region, we measured the luciferase reporter gene (y-axis) driven by humanRPS19 in which we deleted the binding motif for potential transcription factors c-Rel/Rel-A (Del κB element, clones 1 and 2). Unexpectedly, the luciferase reporter gene activity was not decreased but in fact increased 3-fold compared with the wild-typeRPS19 gene. Data from individual experiments are shown.

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