The fruit fly, Drosophila melanogaster, produces juvenile hormone (JH) to assist in development through different stages of metamorphosis and also in reproduction in adult flies. Juvenile hormone has been shown to induce and regulate expression of certain genes. Here, using Drosophila S2 cells treated with methoprene, a juvenile hormone analog, I attempted to show induced expression of two genes, dRNase Z and Dacapo. Utilizing reverse transcriptase-polymerase chain reaction, both genes were identified via RNA. However, the results obtained were insufficient to describe a quantitative increase in gene expression as a result of methoprene treatment.
Figure 1-Schematic diagram of the location of primers used in RT-PCR to amplify Dacapo and dRNase Z
Figure 2-RT-PCR amplification of Dacapo and dRNase Z genes using specific primers. Cells were either treated with methoprene or not treated. RP49 was used as the loading control
Figure 3-Partial Sequence Alignment of purified PCR Product with reported Dacapo Sequence from BLAST 2 Analysis
Figure 4-Partial Sequence Alignment of PCR Product with reported dRNase Z Sequence from BLAST 2 Analysis
Based on the primers designed and used in these experiments, the products of Reverse Transcriptase-PCR corresponded to the predicted fragment sizes:
dRNase Z: 202bp
Sequencing results on these products followed by BLAST 2 sequence analyses further confirmed the identities of dacapo and dRNase Z
The results of RT-PCR and sequencing showed that the primers designed were able to amplify dRNase Z and dacapo
These results, however, were insufficient for quantitation
For more conclusive results, further analysis needs to be performed on the effect of methoprene on S2 cells, possibly Real Time-PCR, which would quantify any increases in gene induction that might be seen in the methoprene-treated cells as compared to untreated cells
Another suggestion is to treat the cells with actual juvenile hormone. This would be more tedious since JH degrades rapidly but any results obtained might be significant and more reliable
I would like to thank Travis Bernardo, Dr. Edward Dubrovsky and Dr. Veronica Dubrovskaya for their advice, inspiration and contributions to this project.
I would also like to thank Leleesha Samaraweera and Bo Liu for being perfect guides.
Finally, I would like to thank Dr. Berish Rubin for allowing me the opportunity to undertake a challenging project that could only improve my abilities and make me a more confident individual.
|This document was last modified 05/05/2008.|
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