VIEWS: 0 PAGES: 1 POSTED ON: 3/14/2013
NATIONAL HIGH MAGNETIC FIELD LABORATORY 2005 RESEARCH REPORT EFFECTS OF HIGH MAGNETIC FIELDS ON IN VITRO TRANSCRIPTION Marianna Worczak (UF, Physics via NHMFL REU; and Clarkson University, Chemistry), Kimberly Wadelton (UF, Physics via NHMFL REU; and Sweet Briar College, Physics), James Ch. Davis (UF, Physics), Anna-Lisa Paul (UF, Horticultural Sciences), and Mark W. Meisel (UF, Physics) Introduction The study of biological molecules in high magnetic fields began in the early 1970’s, when major advances in NMR instrumentation allowed researchers to observe the partial alignment of nucleic acids and certain phospholipids. Magnetic effects on protein orientation were originally considered in theoretical calculations of the magnetic anisotropy of peptide bonds [1,2]. The alignment of biological molecules in magnetic fields has raised the question of how biological processes might be affected by the presence of strong magnetic fields. Work involving exposure of Arabidopsis thaliana to high magnetic fields noted an induction of a gene reporter driven by the alcohol dehydrogenase promoter . This observation suggested that the magnetic field impacts normal biological function, which affected gene expression. Since many reactions and environmental interactions influence gene expression in a living system, our present work investigates in vitro transcription of a single process. Experimental Details Commercially available in vitro transcription kits contain RNA polymerases isolated from bacteriophages. These polymerases, such as T7 and SP6 as used in our work, are single unit globular proteins, which maintain active site structure and function similar to multisubunit polymerases typically found in higher organisms. Since phage polymerases have faster kinetics than their multisubunit homologs, large amounts of RNA can be quickly produced for study. The working hypothesis fueling our experiments was that strong magnetic fields acting on the magnetic anisotropies already present in the RNA polymerases would lead to an alteration in the three dimensional shape of the protein. This alteration, whether it is a partial alignment or substantial distortion, was hypothesized to have some effect on enzymatic function. Results and Discussion To test the hypothesis, experiments employing T7 and SP6 polymerase in vitro transcription kits were preformed in the presence of magnetic fields ranging up to 25 T. Preliminary results analyzed by gel electrophoresis indicate that transcripts were not truncated. However, T7 results suggest a subtle reduction in efficiency with increasing magnetic field, which indicates the potential of some minor effects of the magnetic field on transcription. Results from SP6 indicate an overall decrease in transcript produced for reactions at 9 Tesla but not at 4.5 Tesla. If our results indeed arise from an alteration of polymerase conformation, the observed difference in effects between SP6 and T7 reactions may be due to their slightly different structures. Future tests are necessary to verify the effects observed. In addition, experiments in simple organisms such as bacteria may allow for control of some variables confounding original plant studies, while still providing a system more sensitive to subtle magnetic field effects than detectable with the in vitro kits. Acknowledgements This work was partially supported by the National High Magnetic Field Laboratory (NHMFL) Summer 2005 REU Program at the University of Florida in Gainesville. Work was also supported in part by NSF DMR-0305371 and NASA grant NNA04CC61. We acknowledge R.J. Ferl and W.B. Gurley for enlightening conversations. References  Worcester, D.L., Proc. Nat. Acad. Sci. USA, 75, 5475-5477 (1978).  Pauling, L., Biophysics, 76, 2293-2294 (1979).  Paul, A.-L., et al., in Magnetic Fields: Proceedings of the International Workshop on Materials Analysis and Processing in Magnetic Fields, edited by Hans J. Schneider-Muntau and Hitoshi Wada (World-Scientific, Singapore, 2005) pp. 238-242.
Pages to are hidden for
"NHMFL 2005 Research Report - University of Florida"Please download to view full document