Investigation of Ideal in Vitro Transcription Parameters in Frankia Strain ArI-5
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Authors
Bozyk, Paul Douglas
Issue Date
1995
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
The actinomycete Frankia has nitrogen fixing capabilities either in a
free-living state or in a symbiotic relationship with various genera of
actinorhizal plants in temperate zones. At this time, little work dealing with
the molecular nature of this bacteria has been done. This research was part of
a larger project to define a promoter sequence which can direct transcription
in Frankia cells. Specifically, the purpose of this project is to identify in vitro
transcription parameters with respect to KCI and MgCl2 concentrations that
are most similar to the environment of the cell, and to determine whether
the transcripts formed under such conditions initiate in the same location as
the analogous cellular mRNA's. After lysing the Frankia cells and isolating
their transcriptional extract, in vitro transcription was performed using
radiolabeled 32P-UTP. The level of incorporated radioactivity was measured
with a Scintillation counter and transcripts were identified by
autoradiography of formaldehyde agarose gels. The data suggests that
optimal transcription using the Frankia RNA polymerase occurs at KCI
concentration of 50 mM and MgCl2 concentration of 2 mM. This information
could be useful in the identification of a transcriptional start site and
promoter region for this Frankia transcript using methods of primer
extension and DNA sequencing.
Description
iii, 37 p.
Citation
Publisher
Kalamazoo College
License
U.S. copyright laws protect this material. Commercial use or distribution of this material is not permitted without prior written permission of the copyright holder.