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Identification of promoter elements in 5'-flanking region of murine cyclic nucleotide phosphodiesterase 3B gene

Liu, Hanguan; Tang, Jing Rong; Degerman, Eva LU and Manganiello, Vincent C (2005) In Phosphodiesterase Methods and Protocols (Methods in molecular biology) 307. p.109-124
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Chapter in Book/Report/Conference proceeding
publication status
published
subject
keywords
mutation, electrophoretic mobility shift assay, reverse transcriptase-polymerase chain reaction, promoter, Phosphodiesterase 3B, DNA sequencing, transcription initiation
in
Phosphodiesterase Methods and Protocols (Methods in molecular biology)
volume
307
pages
109 - 124
publisher
Humana Press
external identifiers
  • pmid:15988059
  • scopus:24644520017
ISSN
1064-3745
1940-6029
DOI
10.1385/1-59259-839-0:109
language
English
LU publication?
yes
id
430df004-4fa2-4382-aea5-6b9e675c1cdb (old id 1133638)
date added to LUP
2008-06-17 15:38:20
date last changed
2017-01-01 04:42:38
@inbook{430df004-4fa2-4382-aea5-6b9e675c1cdb,
  abstract     = {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.},
  author       = {Liu, Hanguan and Tang, Jing Rong and Degerman, Eva and Manganiello, Vincent C},
  issn         = {1064-3745},
  keyword      = {mutation,electrophoretic mobility shift assay,reverse transcriptase-polymerase chain reaction,promoter,Phosphodiesterase 3B,DNA sequencing,transcription initiation},
  language     = {eng},
  pages        = {109--124},
  publisher    = {Humana Press},
  series       = {Phosphodiesterase Methods and Protocols (Methods in molecular biology)},
  title        = {Identification of promoter elements in 5'-flanking region of murine cyclic nucleotide phosphodiesterase 3B gene},
  url          = {http://dx.doi.org/10.1385/1-59259-839-0:109},
  volume       = {307},
  year         = {2005},
}