We describe techniques for identifying functional promoter elements in the 5'-flanking region of the murine cyclic nucleotide phosphodiesterase 3B (mPDE3B) gene. The 5'-flanking region of the mPDE3B gene was cloned and sequenced, and putative transcription factor binding sites were identified with computational tools. A series of reporter plasmids containing the luciferase gene fused to different fragments of the 5'-flanking region of the mPDE3B gene was constructed and used to transfect 3T3-L1 fibroblasts or differentiating adipocytes. Reporter gene assays showed that there are two promoter regions in the 5'-flanking region in the mPDE3B gene: a distal region located approx 4 kb upstream of the translation initiation site that contains cAMP-response element (CRE) cis-acting elements, and a proximal region that is GC rich and lacks TATA sequences. The distal promoter region induced much higher luciferase activity than did the proximal one. Mutation of the CRE sequences or reversal of the orientation of the CRE-containing region abolished promoter activity of the distal region. Electrophoretic mobility shift assay analysis indicated that binding to CRE elements was greater in nuclear extracts from differentiating adipocytes than from fibroblasts. Mapping of transcription initiation sites suggested that the distal promoter region might function as an enhancer, whereas the proximal promoter drives transcription of the mPDE3B gene.
|Research areas and keywords
- Endocrinology and Diabetes
- mutation, electrophoretic mobility shift assay, reverse transcriptase-polymerase chain reaction, promoter, Phosphodiesterase 3B, DNA sequencing, transcription initiation
|Title of host publication||Phosphodiesterase Methods and Protocols (Methods in molecular biology)|
|Publication status||Published - 2005|