Synthetic Promoter Elements Obtained by Nucleotide Sequence Variation and Selection for Activity

Eukaryotic transcriptional regulation in different cells involves large numbers and arrangements of cis and trans elements. To survey the number of cis regulatory elements that are active in different contexts, we have devised a high-throughput selection procedure permitting synthesis of active cis...

Full description

Saved in:
Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2000-03, Vol.97 (7), p.3038-3043
Main Authors: Edelman, Gerald M., Meech, Robyn, Owens, Geoffrey C., Jones, Frederick S.
Format: Article
Language:English
Subjects:
Base Sequence
Biochemistry
Biological Sciences
Cell lines
Deoxyribonucleic acid
DNA
DNA - genetics
DNA-Binding Proteins - genetics
Genetic mutation
Genetic Vectors
Libraries
Molecular Sequence Data
Neurons
Plasmids
Polymerase chain reaction
Promoter Regions, Genetic
Proteins
Retroviridae - genetics
Stem cells
Transfection
Virus Assembly
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Eukaryotic transcriptional regulation in different cells involves large numbers and arrangements of cis and trans elements. To survey the number of cis regulatory elements that are active in different contexts, we have devised a high-throughput selection procedure permitting synthesis of active cis motifs that enhance the activity of a minimal promoter. This synthetic promoter construction method (SPCM) was used to identify >100 DNA sequences that showed increased promoter activity in the neuroblastoma cell line Neuro2A. After determining DNA sequences of selected synthetic promoters, database searches for known elements revealed a predominance of eight motifs: AP2, CEBP, GRE, Ebox, ETS, CREB, AP1, and SP1/MAZ. The most active of the selected synthetic promoters contain composites of a number of these motifs. Assays of DNA binding and promoter activity of three exemplary motifs (ETS, CREB, and SP1/MAZ) were used to prove the effectiveness of SPCM in uncovering active sequences. Up to 10% of 133 selected active sequences had no match in currently available databases, raising the possibility that new motifs and transcriptional regulatory proteins to which they bind may be revealed by SPCM. The method may find uses in constructing databases of active cis motifs, in diagnostics, and in gene therapy.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.040569897