Expression of dRNase Z and Dacapo mRNA in Drosophila melanogaster cells exposed to Juvenile Hormone analog




Cyntra Singh

Introduction

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.

Juvenile hormone (JH) is a sesqueterpenoid compound produced by many insect species to aid in several key processes during development and maturation. JH plays important roles during the molting pupal and adult stages in Drosophila. Reproductively, JH is necessary for ovarian maturation in females and overall reproductive maturation in males (Riddiford 2008). The JH synthetic analog, methoprene, has been reported to increase expression of various genes in the fruit fly, including juvenile hormone induced-1 (JhI-1), and juvenile hormone induced-26 (JhI-26), as described by Dubrovsky et al in 2000. More recent studies have identified JhI-1 as a tRNA 3 processing gene, dRNase Z (Dubrovsky et al 2004).

During the translational phase of protein biosynthesis, tRNA molecules transfer specific amino acids to the growing polypeptide chain at the ribosomal site. For maturation of the tRNA molecule, additional 5 and 3 sequences that are transcribed with the entire molecule must be removed by enzymes. RNase P enzyme removes the 5 sequence and the endonuclease RNase Z removes the 3 sequence. RNase Z, belongs to the human ELAC1/ELAC2 group of conserved proteins (Dubrovsky et al 2004).

Linkage analysis and positional cloning described ELAC2 (also hereditary prostate cancer 2 gene, HPC2) as the candidate susceptibility gene for prostate cancer in human males (Xu et al 2001). The presence of missense mutations was described as a factor in increased incidences of prostate cancer (Rebbeck et al 2000). Further analysis into the functioning of ELAC2, revealed a downstream signaling cascade involving ELAC2, transforming growth factor-β (TGF-β), Smad proteins, and cyclin-dependent kinase (CDK) inhibitors including p21 and p15. All of these proteins are involved in regulation of the cell cycle, controlling proliferation, and growth and differentiation to various degrees. ELAC2 induces the TGF-β/Smad cascade involving the signal transducer, FAST-1 and the transcription factor, Sp1. This chain of events further activates p21 transcription, thereby causing cell growth inhibition through the cyclin-dependent kinase inhibitor protein, p21 (Noda et al 2006).

In Drosophila melanogaster, the ELAC2 ortholog is dRNase Z, which is encoded by JhI-1 (Dubrovsky et al 2004). The Drosophila ortholog of the p21 cyclin-dependent kinase inhibitor was identified as Dacapo. Its significance to the fruit fly involves the endocycle process, whereby cells engage in DNA replication in the absence of mitosis (Hong et al 2007).

Because of the relationship between ELAC2 and p21, and their proposed involvement in increased prostate cancer risk, I attempted to identify the Drosophila orthologs of both genes (dRNase Z and Dacapo), as being expressed via induction by the juvenile hormone analog, methoprene. My purpose was to describe the potential increase in dRNase Z expression and consequently, dacapo expression as a result of methoprene treatment. If successful, further studies could be done using Drosophila as a viable model.

Figures


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:
Dacapo: 171bp
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


Acknowledgments

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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