Figure 1- Total RNA extracted from HeLa cells. RNA was extracted according to Materials and Methods. RNA products were visualized by ethidium bromide staining on an agarose gel. 100 bp ladder (Lane 1) and RNA (Lane2).
Figure 2- DNase treated RNA product. DNase treatment was performed according to Materials and Methods. 100 bp ladder (Lane 1) and DNased treated RNA.(Lane 2).
Figure 3- RT-PCR amplification of mtTFA. PCR amplification was performed according to Materials and Methods. Actin and mtTFA were amplified and fractionated on an agarose gel (lanes 3 and 7 and lanes 4 and 9 respectively). Lanes 2 and 7 represent the negative control using the mtTFA primers where no DNA was added to the sample. Lanes 1 and 6 represent a 100bp ladder.
RNA extracted from HeLa cells and run on a 0.8% agarose gel revealed the presence of 3 distinct products (Figure 1). The highest MW product is DNA. The middle sized product is 28S rRNA and the smallest product is 18S rRNA. After DNase treatment (Figure 2), the DNA band has been eliminated. The 28S rRNA and 18S rRNA bands are still present. The isolated RNA was subjected to reverse transcription. The cDNA product was PCR amplified using primers mtTFA1 and mtTFA2 or actin primers. Actin was used as a positive control. Actin migrated correctly and is approximately 700 bp in length (Figure 3: Lanes 3 and 8). mtTFA DNA migrated to an approximate position of 750 bp (Figure 3: Lanes 4 and 9). Sequencing was performed as described in Materials and Methods and the derived nucleotide sequence was aligned with the human mitochondrial transcription factor 1 mRNA from GenBank. 722 bp of the reported 750 bp were sequenced with 99% homology. But within the readable sequence there was 100% homology to the reported sequence.
mtTFA RNA isolated and characterized as described above has high homology with the previously published sequence. This suggests the techniques used can properly extract, isolate and amplify mtTFA RNA or any mRNA.
Further studies have been done on mtTFA and its role in oxidative phosphorylation, embryogenesis in mice, spermatogenesis in humans and respiratory chain defects in humans. The sequence of mtTFA was discerned and checked for mutations is a study of early-onset encephalomyopathy (EOE). EOE is associated with tissue specific mitochondrial DNA depletion, however, no changes from the published sequence were observed. The sequence of mtTFA was also used to study hyperfunctional parathyroids. Again, changes were not observed from the published sequence. mtTFA is quite important to normal cellular function through the replication and transcription of mtDNA. Although changes in the sequence have not been observed in studies of EOE and hyperfunctional parathyroids, changes might be observed in other mtDNA deficient disorders. However, it seems that the expression of mtTFA is an important factor. Its expression has been observed to increase in chronic liver disease and has been shown to be suppressed by IFN-alpha and IFN-gamma and is believed to be a pathway through which IFNs depress mitochondrial respiration. The approach described in this paper allows for the ready characterization of the mRNA sequence of mtTFA.
The author would like to acknowledge the unending support of Sabrina Volpi and Rocco Coli, without whose support this project could not have been completed. The author also thanks Dr. Berish Y. Rubin for all his help and support with the project and its exposure.
|This document was last modified 01/31/2006.|
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