词条 | Promoter bashing |
释义 |
Promoter bashing is often done with deletions from either the 5' or 3' end of the DNA strand; this assay is easier to perform based on repeated restriction digestion and gel-purifying fragments of specific sizes. It is often easiest to ligate the promoter into the reporter, generate a large amount of the reporter construct using PCR or growth in bacteria, and then perform serial restriction digests on this sample. The ability of upstream promoters can be easily assayed by removing segments from the 5' end, and the same for the 3' end of the strand for downstream promoters.[4] 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] ProcedureThis is an example procedure for a promoter bashing assay, adapted from Boulin et al.:[6]
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
References1. ^Kamvysselis, M. (2003). Computational molecular genomics: genes, regulation, evolution. (Doctoral Dissertation). Retrieved from http://web.mit.edu/manoli/www/thesis/Intro.html {{molecular genetics methods}}2. ^Chalfie, M., & Kain, S. (2005) Methods of Biological Analyses, Green Fluorescent Protein: Properties, Applications, and Protocols. Wiley. 3. ^Matsukura, S., Stellato, C., Plitt, J. R., Bickel, C., Miura, K., Georas, S. N., Casolaro, V., Schleimer, R. P. (1999). "Activation of Eotaxin Gene Transcription by NF-κB and STAT6 in Human Airway Epithelial Cells". J Immunol 163:6876-6883. PMID [https://www.ncbi.nlm.nih.gov/pubmed/10586089 10586089]. 4. ^Engstrom, E. M., Izhaki, A., Bowman, J. L. (2004). "Promoter bashing, microRNAs, and Knox genes. New insights, regulators, and targets-of-regulation in the establishment of lateral organ polarity in Arabidopsis." Plant Phisiol 135(2): 685-94. doi: [https://dx.doi.org/10.1104/pp.104.040394 10.1104/pp.104.040394]. PMID [https://www.ncbi.nlm.nih.gov/pubmed/15208415 15208415]. PMC [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC514105/ 514105]. 5. ^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:[https://dx.doi.org/10.1038/sj.cr.7290136 10.1038/sj.cr.7290136]. 6. ^Boulin, T. et al. Reporter gene fusions (April 5, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi [https://dx.doi.org/10.1895/wormbook.1.106.1 10.1895/wormbook.1.106.1], http://www.wormbook.org. 2 : Molecular biology techniques|Molecular genetics |
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