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RNA Editing in the cacophony Ion Channel
Jason Bannock, Barry Hoopengardner
Department of Biomolecular Sciences, Central Connecticut State University
The following is an example of Adenosine to inosine RNA editing. This
INTRODUCTION
edit was from the Slowpoke ion channel of Drosophila melanogaster
A-to-I RNA editing is a modification process in which an Adenosine is edited to an Inosine post-transcriptionally by the ADAR family of enzymes. ADARs are adenosine
using the 20Useq, and 24Dseq2 primer combination. The sequence was
deaminases that act on RNA. At this time we are using known RNA editing sites in the fruit fly (Drosophila melanogaster), and the honey bee (Apis mellifera), and trying
the result of a PCR from adult flies ranging in age from 1-3 days old. The
to gather comparative data from the common carpenter ant (Camponotus pennsylvanicus), and the red harvester ant (Pogonomyrmex barbatus ). Our primary target for
relative peak strength shown by the arrow suggests an approximate
locating editing sites is in a calcium channel called cacophony, however the primary ion channel being used for the determination of RNA editing frequency is a
editing frequency of 50 percent.
potassium channel known as Slowpoke. The ultimate goal of this research is to utilize the knowledge gained on placement and function, as well as the relative
frequencies of the editing sites, and use it to produce pesticides that act against agricultural pests and at the same time do no harm to the pollinators.
To this point there has not been any work to show the levels of RNA editing throughout the adult life span of an organism, until a former colleague, James Arnone
was working on a potassium channel (Slowpoke) using restriction enzyme digests of a time course of adult Drosophila melanogaster and had data to suggest that the
levels of editing did not change over time. With this new data the focus of the research has been shifted to showing concretely that the frequency of RNA editing,
much like many other gene regulation mechanisms, does not change over the adult life span.
PRELIMINARY DATA
Using primers designed to amplify a section of the Slowpoke Potassium
This Gel is demonstrating the first step in the “aging” project. It was used to examine any possible
This gel is showing the consistency of our data. The consistency is shown here because
channel in Drosophila melanogaster, we have shown the possible
problems with our primer combinations or RNA. From this data we concluded that there was
the last four lanes of the gel all use the “Honing” forward primer and “Biene” reverse
presence of the ion channel in Apis mellifera. As you can tell, if in fact
nothing wrong with the Cac Cluster primer combination, used for the cacophony ion channel. This
primer; however they are from two RNA isolations from which I ran four reverse
the amplified band in the fourth lane of the gel is the Slowpoke ion
conclusion was drawn from the 8 positive lanes. Another conclusion drawn from this data was
transcriptions (two form each isolation) and four PCR reactions (one form each RT). The
channel, it is not 100 percent conserved however, a band does appear
that there was a problem with either the RNA isolation step or the Reverse Transcription step. At
second interesting thing to notice with this data is that all four samples that used the
suggesting a certain level of conservation in the ion channel between the
this time the problem can not be diagnosed any further without additional experiments. This
Honing/Biene primers that were degeneratively designed to work with the honey bee, Apis
fruit fly and the honey bee. At this time we are attempting to gather more
experiment was also performed for the two regions of interest in the Slowpoke ion channel (data
mellifera, are actually working on Camponotus pennsylvanicus, the common carpenter
data supporting our results, including; gel electrophoresis data, and
not shown).
ant.
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Honing/Biene (8/8 –
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Slowpoke Primers 20useq/24dseq2 (8/31- BEE)
Cac Cluster F1/R1 (8/31- BEE)
Honing/Biene (8/31 - BEE)
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Future Directions: The future goals, in terms of the “pesticide project” are to use determine if there are differences in ion channel editing between agriculturally important insects, this is done with comparative analysis of RT-PCR data and DNA sequencing. The
next step would be to determine the amino acid change due to editing. The amino acid alteration(s) caused by species specific RNA editing, may change features of the cacophony ion channel, which can be targeted by pesticides. The future directions for the
aging project are to use restriction enzyme digests, and densitometry to examine the cacophony ion channel to examine the frequency, and levels of RNA editing. Although I have split the two projects for descriptive purposes they are very much linked to one
another. What I mean by this is that if I observe a 20 percent occurrence of edited transcripts, that target is not a reasonable one for pesticide development, however if I observe a 75 percent or higher frequency than that would be a logical target to start
characterizing. Therefore my project will be aimed at looking at the frequencies in which editing occurs, and picking out possible targets for future research.