140 likes | 277 Views
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
E N D
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
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
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
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
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
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
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
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
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.)
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)
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
General DNA Structure www.emc.maricopa.edu/.../ farabee/BIOBK/BP2.gif
Transcription Activation: Transcription Factors http://life.nthu.edu.tw/~lslpc/StrucBio/chapter9/Fig9-02.jpg