Investigation of Ideal in Vitro Transcription Parameters in Frankia Strain ArI-5
Bozyk, Paul Douglas
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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.