Amplification and sequencing of transcript encoding the shaker homologue in Ixodes Scapularis




Joseph A. Frezzo

Introduction

The shaker gene, or voltage gated potassium channel gene in mammals, represents a highly conserved ancient protein family discovered in most organisms and predicted to be present in almost every organism. The diversity of these channels is so expansive in humans, C. elegans, and Drosophila that each organism contains between 30-100 potassium channels. The potassium channels are divided into two subsets: voltage gated and resting channels. Voltage gated potassium channels are six pass transmembrane proteins with remarkable specificity for a single ion that are only activated upon membrane potential fluctuations.
The prevalence of the voltage gated potassium channels in all organisms, and their high homology allows for reliable detection of homologues from various organisms whose genes have not been sequenced. Here is provided the experimental approach to amplification and sequencing of partial transcripts of Ixodes scapularis shaker like homologue. The mRNA sequences for shaker from the three related arthropods Drosophila melanogaster, Anopheles gambiae, and Apis mellifera were aligned and primers were designed to the most highly conserved regions, but wholly specific to Drosophila melanogaster. Primers were designed around the S5 segment, specifically to exon 10, and also in regions of exons 3 and 4. Sequence homology was then studied for each region.

Figures


Figure 1-Figure 1. PCR products using primers 101A & 102B and 31A & 32B. Panel A: Exon 3 amplicons for tick (IS) and Drosophila (DM) with a non template control (NTC). Panel B: Exon 10 amplicons from tick (IS), Drosophila (DM) and non template control (NTC). The sizes of both sets of amplicons is ~100 bp. Size comparison was done using a 100 bp ladder (L)


Figure 2-Figure 2. PCR products using primers 42F and 41R. Sizes of PCR products are determined by comparing the size with that of the 100 bp ladder. Lane 1 presents the PCR products generated from the purified Tick genomic DNA. The product at 150 bp is the expected size while the 100 bp product was determined to be nonspecific. Lane 2 presents the product generated from the purified Drosophila genomic DNA which is of the expected size. lane 3 is the non template control


Figure 3-Figure 3. Sequence homlogy studies were conducted on each tick PCR product and the published Drosophila sequence. the alignments reveal 100% sequence homology for exon 3, 97% sequence homology for exon 4 and 98% sequence homology for exon 10.


The National Institute of Allergy and Infectious Diseases the DNA sequencing of the deer tick in hopes of understanding the role ticks play in passing pathogens to humans that cause lyme disease, rocky mountain spotted fever and tularemia. The research project undertaken provides an easy and efficient means to begin the DNA sequencing on a small scale which can be done in almost any molecular biology and genetics laboratory.
The sequence homology between all organisms for the voltage gated potassium channels provided easy primer design. Since the gene was undoubtedly expected to be present in the tick, the project easily provided a partial sequence of the voltage gated potassium channel.

Full Paper

Acknowledgments

I would like to thank Dr. Sylvia Anderson, Jinsong Qiu, Dr. Berish Rubin and Lisa Sarran. I would also like to thank the department of biological sciences for funding this amazing project.


This document was last modified 05/17/2006.
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