Promoter bashing
Promoter bashing is a technique used in
Promoter bashing is often done with deletions from either the
As the promoter commonly contains binding sequences for proteins affecting transcription, those proteins are also necessary when testing the effects of the promoter. Proteins which associate with the promoter can be identified using an electrophoretic mobility shift assay (EMSA), and the effects of inclusion or exclusion of the proteins with the mutagenized promoters can be assessed in the assay. This allows the use of promoter bashing to not only discover the location on the DNA strand which affects transcription, but also the proteins which affect that strand. The effects of protein interactions with each other as well as the binding sites can also be assayed in this way; candidate proteins must instead be identified by protein/protein interaction assays instead of an EMSA.[5]
Procedure
This is an example procedure for a promoter bashing assay, adapted from Boulin et al.:[6]
- Clone the region of DNA thought to act as a promoter. Cloning is necessary for the assay because it ensures that the promoter is the only factor affecting expression. This step often involves extraction of the DNA from the organism it resides in and PCR amplification.
- Sequence the region. DNA Sequencingis necessary to identify differences in mutated promoters from the wild-type promoter, and to correlate those differences with differences in gene expression. Additionally, it helps with the restriction digest of the region.
- Digest with appropriate restriction endonucleases. The region can be digested to remove elements which are thought to not be part of the promoter. Additionally, the reporter gene must be inserted a set distance from the promoter for most promoters. In some methods of promoter bashing, multiple restriction digests are used to systematically remove elements of the promoters—this method ensures that the regions of the promoter removed do not contribute to reporter expression.
- Mutagenize the promoter. Mutating the promoter is necessary if the method of removing part of the promoter with restriction digestion is not used. Many mutated strands can be generated, and the strands sequenced and the activities of the promoters assayed. This is often necessary because one mutation cannot be guaranteed to inactivate a binding site. Non-directed PCR-based mutagenesis can also be used; the parameters of the mutagenic PCR reaction can be adjusted to introduce a reasonable number of mutations. However, the random nature of PCR requires that more strands are assayed downstream of this step.
- Ligate to reporter gene. The promoters to be assayed must be ligated to a reporter gene so that gene expression levels can be measured. The reporter gene must be a sufficient distance from the promoter that the promoter affects it as a wild-type promoter would affect a gene. This can be verified with the positive control (full promoter).
- Transform cells of interest with the various promoter:reporter constructs. The promoter and reporter constructs must be ligated into a plasmid and transformed into cells in which that plasmid can be expressed to measure the activity of each promoter sequence. Proteins which affect the promoter must also be added to those cells — often those proteins are placed on the same or different plasmid under the regulation of a constitutively active promoter.
- Measure reporter-gene transcription rates. The gene products are assayed and the rates of reporter transcription are measured.
From the data received from assaying the different promoters, the effects of various parts of the promoter can be ascertained. However, it is possible that there may not be enough data present and the assay must be re-run with a different promoter region and/or different mutations.
See also
References
- ^ Kamvysselis, M. (2003). Computational molecular genomics: genes, regulation, evolution. (Doctoral Dissertation). Retrieved from http://web.mit.edu/manoli/www/thesis/Intro.html
- ^ Chalfie, M., & Kain, S. (2005) Methods of Biological Analyses, Green Fluorescent Protein: Properties, Applications, and Protocols. Wiley.
- PMID 10586089.
- .
- ^ Guo, J. Y., Xu, J., Mao, D. Q., Fu, L. L., Gu, J. R., Zhu, J. D. (2002). "The promoter analysis of the human C17orf25 gene, a novel chromosome 17p13.3 gene". Cell Research 12:339-352. doi:10.1038/sj.cr.7290136.
- ^ Boulin, T. et al. Reporter gene fusions (April 5, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi 10.1895/wormbook.1.106.1, http://www.wormbook.org.