Glutathione peroxidases (GPXs) are a family of enzymes in both animal and plant kingdoms that reduce reactive oxygen species (ROS). Specifically, GPXs reduce H2O2, organic hydroperoxides and lipid peroxides, protecting cells from oxidative damage.
Figure 1-RT-PCR products.
Figure 2-H. annuus cDNA from haGPX-2 and the 200 bp mRNA RT-PCR product from Xanthium. GXCF/R primer positions are indicated by arrows and intron location with a vertical line.
Figure 3-Genomic DNA and mRNA alignment. Gray strand is the genomic DNA, and light blue strand, above, is the mRNA sequence, (….) imply sequences that could not be read.
The GXCF/R primers, which recognize highly conserved regions in GPX transcripts, amplified two bands, 200 and 600 bp. The GX1F/R primers, which were designed to the 5' end of the mRNA transcript, did not amplify any mRNA. This region of GPX is not a conserved region and perhaps the homology between H. annuus and Xanthium was too low. β-actin was used as a control, and its primers amplified three bands - most likely caused by the low annealing temperatures used. The identity of these bands remains unknown.
The 200 and 600 bp RT-PCR products were sequenced. The 200 bp product is a Xanthium mRNA transcript for mRNA (Figure 2). 93.75% of readable sequence is homologous to the H. annuus mRNA, and most of the discrepancies are transitional base changes. The 600 bp product is most likely genomic DNA containing an intron. A second explanation that the 600 bp product is another mRNA caused by alternative splicing is unlikely due to the nature of the 600 bp sequence. Long sequences of A’s and T’s prevented the intron from being sequenced entirely and only a partial intron sequence is proposed (Figure 3). The proposed intron contains a 5′ GT donor site and a 3′ AG acceptor splice site, and is AT rich (65.6%).
In future research RACE will be used to sequence entire mRNA transcript. The RT-PCR products will be cloned for sequencing, and the proposed intron will be searched for stop codons. Lastly, GPX expression experiments will be performed using primers that will only amplify mRNA.
I would like to thank JinSong Qui and Brian Fox for their support and invaluable advice and Dr. Lewis for always being willing to toss around ideas with me. I would like to thank Dr. Rubin for his guidance, his lab for providing me with necessary lab space, Dr. Anderson for synthesizing my primers and Fordham University for funding this research.
|This document was last modified 01/31/2006.|
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