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Effects of High Magnetic Fields on In Vitro Transcription*

Effects of High Magnetic Fields on In Vitro Transcription*. Marianna Worczak Clarkson University Kim Wadelton Sweet Briar College James Ch. Davis Department of Physics, University of Florida Anna-Lisa Paul Horticulture and the Biotechnology Program, University of Florida Mark W. Meisel

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Effects of High Magnetic Fields on In Vitro Transcription*

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  1. Effects of High Magnetic Fields on In Vitro Transcription* Marianna Worczak Clarkson University Kim Wadelton Sweet Briar College James Ch. Davis Department of Physics, University of Florida Anna-Lisa Paul Horticulture and the Biotechnology Program, University of Florida Mark W. Meisel Department of Physics, University of Florida *Work part of the NHMFL Summer 2005 REU Program

  2. Introduction and Motivation • Work by Paul et al (to be published) growing plants in high magnetic fields showed a genetic stress response • 1970’s NMR studies suggested magnetic effects on biomolecules • Hypothesis: Field disrupts or alters prominent cell processes • Transcription is first step of all gene expression • Transcription is constantly active in living organisms • In vitro transcription: transcription in a test tube Arabidopsis thaliana

  3. Transcription: The beginning of Gene Expression • Starts Gene Expression • Triplet of DNA base pairs (ATCG) code for protein • Polymerase makes RNA transcript with complimentary base pairs to DNA template (UAGC) • Transcript used in translation to make protein • Proteins perform cell functions http://oregonstate.edu/instruction/bb451/winter2005/stryer/ch28/Slide9.jpg

  4. Methods: In Vitro Transcription • In Vitro involves a cell-less environment • Ribomax T7 Express and Ribomax SP6 kits purchased from Promega Corporation • T7 and SP6 are fast acting RNA polymerases from Bacteriophages • Quick reactions, simple protocol • Controlled environment • Control Template makes transcripts of known length for analysis Bacteriophage infecting E.Coli http://www.dform.com/projects/t4/gif/t4.gif

  5. Experiments: T7 and SP6 Reactions • T7 Reactions: • Employed a frozen-start method • Bore control, 4.5 Tesla, 9 Tesla • Times: 1,5, 10, 20 minutes • Room Temperature • SP6 Reactions: • Employed a frozen-start method • Bore control, 4.5 Tesla, 9 Tesla • Times: 2, 60, 90, 120 minutes • Temperature: 37°C

  6. Analysis: Gel Electrophoresis Example Agarose Gel http://www.dmd.nl/images/MLPA_agaroseDMD.jpg http://www.stanford.edu/group/hopes/diagnsis/gentest/f_s02gelelect.gif

  7. Results: T7 Reactions T7 Electrophoresis Results 4.5 Tesla 1 minute 4.5 Tesla 5 minutes 4.5 Tesla 10 minutes 4.5 Tesla 20 minutes 9 Tesla 1 minute 9 Tesla 10 minutes 9 Tesla 20 minutes Control 1 minute Control 5 minutes Control 10 minutes Control 20 minutes 9 Tesla 5 minutes 2.3kb 1.1kb • Band Intensity quantified with BioRad QuanityOne • Unit less intensities were compared by subtracting background and setting 1 minute reaction as standard • Small overall decrease in rate of transcript production at 4.5 and 9 Tesla

  8. Results: SP6 Reactions Control 60 min. Control 90 min. Control 120 min. 4.5 Tesla 2 min. 4.5 Tesla 60 min. 4.5 Tesla 90 min. 4.5 Tesla 120 min. 9 Tesla 2 min. 9 Tesla 60 min. 9 Tesla 90 min. 9 Tesla 120 min. Control 2 min. • Reactions produced large amounts of transcripts after 60 minutes • 9 Tesla reactions appear to produce less transcripts • Intensities were not quantified since visual inspection did not display a trend as in T7 1.8kb • Unlike T7, the tertiary structure of SP6 is not known • SP6 has 874 amino acids compared with 883 in T7 • A difference in structure may lead to different effects of the magnetic field

  9. Conclusions • Preliminary T7 results suggest an delay in peak transcript production rate in reactions up to 9 Tesla • Preliminary SP6 results suggest decrease in total transcript produced at 9 Tesla but not at 4.5 Tesla • Differences in structure between T7 and SP6 may explain differences in effect trends RasMol Model T7 RNA Polymerase colored by functional sections. PDB model QLN1 (Tahir et al.)

  10. Future Directions • Analyze Data from 20 and 25 Tesla • Theoretical Calculations by Wadelton et al. suggests model for effects of field on T7 • Investigate Mistranscription • Examine other processes such as replication of Plasmids in Bacteria NHMFL Facility in Tallahassee (http://nmr.magnet.fsu.edu/welcome/users.htm)

  11. Effects of High Magnetic Fields on In Vitro Transcription* Thank You! Questions? Marianna Worczak Clarkson University Kim Wadelton Sweet Briar College James Ch. Davis Department of Physics, University of Florida Anna-Lisa Paul Horticulture and the Biotechnology Program, University of Florida Mark W. Meisel Department of Physics, University of Florida *Work part of the NHMFL Summer 2005 REU Program

  12. General DNA Structure www.emc.maricopa.edu/.../ farabee/BIOBK/BP2.gif

  13. Comparison of DNA and RNA

  14. Transcription Activation: Transcription Factors http://life.nthu.edu.tw/~lslpc/StrucBio/chapter9/Fig9-02.jpg

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