Fig. 1.
Fig. 1. Deletion and point mutation analysis of the CD99 gene promoter region. / (Top) Linear diagram of the promoter region is shown. Putative SP1-binding sites, core sequences (GGGCGG, complement CCGCCC, or both) and the decanucleotide (GGGGCGGGGC) consensus sequences, are located at −274, −242, −151, −95, −48, −5, +24, and +74 and are indicated as black and gray rectangles, respectively. Nucleotide numbers are relative to the transcription start site (+1) published previously14 and are indicated by the arrowhead. (Bottom) Various promoter-luciferase fusion constructs and the promoterless vector as a negative control were transiently transfected into 293 cells. Eighteen CD99 promoter variants 8 5′ deletion derivatives, 4 internal deletion and 4 point mutation derivatives, and 2 3′ deletion derivatives. (A) Relative activities of the 5′ deletion derivatives of the CD99 promoter region. Plasmid names of eight 5′ deletion derivatives and the full-length CD99 promoter construct are listed to the left of the diagram in the middle. The included CD99 promoter region is denoted in the name of each construct. Relative luciferase activity, shown to the right of each construct, is expressed as the -fold of the luciferase activities obtained from the promoter-luciferase fusion construct over the promoterless vector, p(0)luc. To normalize plate-to-plate variations, the construct containing the TK promoter-driven Renilla luciferase (pRLTK) was cotransfected, and the activities of firefly and Renillaluciferases were measured sequentially from a single sample using a dual-luciferase reporter assay system. Data represent averages from 3 separate transfections performed with each construct. (B) Relative activities of the internal deletion and the point mutation derivatives of the CD99 promoter region. The name of internal deletion mutant indicates the position of the deleted region. p1mGC, p2mGC, p3mGC, and p123mGC contain the mutated GC boxes at −95, −48, −5, and −95/−48/−5, respectively. (C) Relative activities of the 3′ deletion derivatives of the CD99 promoter region. The included CD99 promoter region is denoted in the name of each construct. Transfection and analysis were performed as previously described in panel A.

Deletion and point mutation analysis of the CD99 gene promoter region.

(Top) Linear diagram of the promoter region is shown. Putative SP1-binding sites, core sequences (GGGCGG, complement CCGCCC, or both) and the decanucleotide (GGGGCGGGGC) consensus sequences, are located at −274, −242, −151, −95, −48, −5, +24, and +74 and are indicated as black and gray rectangles, respectively. Nucleotide numbers are relative to the transcription start site (+1) published previously14 and are indicated by the arrowhead. (Bottom) Various promoter-luciferase fusion constructs and the promoterless vector as a negative control were transiently transfected into 293 cells. Eighteen CD99 promoter variants 8 5′ deletion derivatives, 4 internal deletion and 4 point mutation derivatives, and 2 3′ deletion derivatives. (A) Relative activities of the 5′ deletion derivatives of the CD99 promoter region. Plasmid names of eight 5′ deletion derivatives and the full-length CD99 promoter construct are listed to the left of the diagram in the middle. The included CD99 promoter region is denoted in the name of each construct. Relative luciferase activity, shown to the right of each construct, is expressed as the -fold of the luciferase activities obtained from the promoter-luciferase fusion construct over the promoterless vector, p(0)luc. To normalize plate-to-plate variations, the construct containing the TK promoter-driven Renilla luciferase (pRLTK) was cotransfected, and the activities of firefly and Renillaluciferases were measured sequentially from a single sample using a dual-luciferase reporter assay system. Data represent averages from 3 separate transfections performed with each construct. (B) Relative activities of the internal deletion and the point mutation derivatives of the CD99 promoter region. The name of internal deletion mutant indicates the position of the deleted region. p1mGC, p2mGC, p3mGC, and p123mGC contain the mutated GC boxes at −95, −48, −5, and −95/−48/−5, respectively. (C) Relative activities of the 3′ deletion derivatives of the CD99 promoter region. The included CD99 promoter region is denoted in the name of each construct. Transfection and analysis were performed as previously described in panel A.

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