Effects of High Magnetic Fields on in vitro Transcription of T7 and ...

<strong>Effects</strong> <strong>of</strong> <strong>High</strong> <strong>Magnetic</strong> <strong>Fields</strong> on in vitro
Transcription <strong>of</strong> T7 and SP6 RNA Polymerases*
Marianna Worczak † , Kimberly Wadelton † ,
James Ch. Davis, and Mark W. Meisel
Department <strong>of</strong> Physics and NHMFL, University <strong>of</strong> Florida
Anna-Lisa Paul and Robert J. Ferl
Department <strong>of</strong> Horticultural Sciences, University <strong>of</strong> Florida
Motivation via work on whole plants
The “Working” Hypothesis
Transcription and T7 Structure
Results with T7 and SP6
Future Directions
* Supported, in part, by the NSF via the NHMFL, DMR-0305371 (MWM),
and NASA grant NNA04CC61 (ALP and RJF).
† NHMFL Research for Undergraduates (REU) Summer 2005.

TAGES - Plant Biomonitors <strong>of</strong> Microgravity
An Etruscan deity who
possesses wisdom. He
appeared from a groove
when a field was newly
ploughed and taught the
gathered Etruscans the
skills <strong>of</strong> divination and
augury.
http://www.christusrex.org/www1/vaticano/ET2-Etrusco.html
Transgenic
Arabidopsis
Gene
Expression
System
• Why Arabidopsis?
"Arabidopsis thaliana: A Model Plant for
Genome Analysis", D.W. Meinke et al.,
Science 282 (1998) 662-682.
• Why Adh?
Questions for the STS-93 experiment:
Is there sufficient hypoxia in Flight to
initiate a hypoxic response?
Is it really hypoxia that the plants see?
Adh-GUS

Adh-GUS Reporter Gene System
Anoxia
Hypoxia
ABA
Cold
Drought
Salt
β-Glucuronidase
- GUS
Adh gene promoter
GUS coding region
Quantification and Localization
(at the level <strong>of</strong> gene expression!)

GUS Activity
[(nmol 4-MUG)/(µg protein)/minute]
70
60
20
10
0
GUS: P = 0.001 (not B or B 2 )
a Leaves (2.5 hr exposure)
Run 1 - NHMFL Cell 5
Run 2 - NHMFL Cell 6
Run 3 - UF Supercon.
Run 4 - NHMFL Cell 5
Log-normal Function
95% Limits
0 5 10 15 20 25
<strong>Magnetic</strong> Field (Tesla)
Real-Time Quantitative PCR
Athb12 Xero2 Xtr7 Cor
Microarray (8k genes)
50
40
30
20
10
0
-10
-20
2.5 hrs Shoot 2.5 hrs Shoot 2.5 hrs Shoot
Control
14 T 21 T
Mag Control Mag 14T Mag 21T
The Quantitative Results (A.-L. Paul et al., MAP1 and to be published.)
[Ikehata et al., J. Appl. Phys. 93 (2003) 6724: yeast in 14 T, 24 hrs: 22 genes ≤ 2 fold changes.]

The “Working” Hypothesis: <strong>Magnetic</strong> Alignment vs. Magnetophoresis
The magnetic energy (Bothner-By, 1996; Tjandra et al., 1997):
E
1
= −
2µ
Variations from anisotropies <strong>of</strong> χ and B:
δE
= −
1
2µ
0
B(
r)
⋅χ⋅B(
r)
An order <strong>of</strong> magnitude comparison <strong>of</strong> the two effects:
Top <strong>of</strong> leaves and Bottom <strong>of</strong> roots:
Anisotropy <strong>of</strong> χ for biomacromolecules:
(Maret, Dransfeld, 1985; Tjandra et al., 1997)
(Valles et al., 1997)
0
{ B(
r)
⋅δχ
⋅ B(
r)
+ 2 B(
r)
⋅ ∆χ
⋅δB(
r)
}
R
≈
2 ∆χ δB
δχ B
(δB/B) ≈ 5 × 10 −3
δχ ≈ 10 −33 m 3 /molecule
∆χ ≈ 10 −29 m 3 /molecule
R < 10 − 2 ⇒ <strong>Magnetic</strong> Alignment > Magnetophoresis !

“Simplicity does not precede complexity,
but follows it.” Alan J. Perlis
(EPIGRAMS IN PROGRAMMING)
Move from “complex” in vivo plants
to a simple in vitro system!
Try in vitro transcription <strong>of</strong> a single process:
Use T7 Ribomax ® Express in vitro Transcription Kit ( ).
Key elements:
1. T7 RNA polymerase is widely studied, including structure.
2. Elements <strong>of</strong> kit are simple and pure (T7 RNA polymerase,
DNA template, rNTPs, and buffer).
3. Protocol simple, and commercial kits available.

http://oregonstate.edu/instruction/bb451/winter2005/stryer/ch28/Slide9.jpg
Transcription
“Fingers”
“Thumb”
“Palm”
“N-Terminal
Domain”
D. Jeruzalmi and T.A. Steitz, EMBO J. 17 (1998) 4101 [PDB 1ARO].

Results: T7 Reactions
T7 Electrophoresis Results
Controls | 4.5 Tesla | 9.0 Tesla
Time (min): 1 5 10 20 | 1 5 10 20 | 1 5 10 20
2.3 kb
1.1 kb
I(t) / I(t = 1 min)
20
15
10
5
0
0 5 10 15 20 25 30
Time (minutes)
Control
4.5 Tesla
9.0 Tesla
20 – 25 Tesla:
“null result”?

Results: SP6 Reactions
SP6 Electrophoresis Results
Controls | 4.5 Tesla | 9.0 Tesla
1.8 kb
Time (min): 1 5 10 20 | 1 5 10 20 | 1 5 10 20
Surprise again: 9 T strong enough to perturb transcription!?
SP6 RNA polymerase structure: not yet determined
(but believed to be similar to T7 RNA polymerase).
SP6: 874 residues
T7: 883 residues

T 7
1QLN: Cheetham and Steitz, Science 286 (1999) 2305.
Model: Theis, Gong, and Martin, Biochemistry 43 (2004) 12709.

<strong>Effects</strong> <strong>of</strong> <strong>High</strong> <strong>Magnetic</strong> <strong>Fields</strong> on in vitro
Transcription <strong>of</strong> T7 and SP6 RNA Polymerases*
Marianna Worczak † , Kimberly Wadelton † ,
James Ch. Davis, and Mark W. Meisel
Department <strong>of</strong> Physics and NHMFL, University <strong>of</strong> Florida
Anna-Lisa Paul and Robert J. Ferl
Department <strong>of</strong> Horticultural Sciences, University <strong>of</strong> Florida
Future Extensions/Work:
T7/SP6: additional data at more/higher magnetic fields & longer time!
(magnetic field power law?)
<strong>Magnetic</strong> Anisotropy from Structure (Worcester, 1978; Pauling, 1979):
calculate/measure/predict differences (via RNCs) ???
(RDC = residual dipolar couplings)
Simple to Complex: E. coli (living system amplifier!) ?
* Supported, in part, by the NSF via the NHMFL, DMR-0305371 (MWM),
and NASA grant NNA04CC61 (ALP and RJF).
† NHMFL Research for Undergraduates (REU) Summer 2005.