Molecular characterization of age-related genes in Drosophila melanogaster

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Tharmarajah, Grace
Quantitative PCR , Gene Expression Analysis , Reverse Transcription Efficiency
Aging, characterized by a time-dependent functional decline, eventually results in the death of an organism. Unfortunately, this complex biological phenomenon is poorly understood. In order to dissect the molecular changes associated with aging, the identification and molecular characterization of the genes that regulate this universal process is absolutely necessary. The expectation is that the isolated genes potentially have human homologues and can be experimentally analyzed in Drosophila melanogaster in order to determine basic function. In an attempt to find candidate genes that may influence aging, the enhancer trap technique was utilized to identify age-related regulatory elements. The genomic regions surrounding the insertion site of the enhancer trap lines have the potential to be regulated by the characterized enhancer. A previous screen determined the temporal pattern of 180 enhancers trap lines, known as DJ lines. Many of these lines demonstrated an expression pattern that was associated with age. Several of the genes within the nearby genomic regions of six sequenced DJ lines, DJ695, DJ710, DJ849, DJ767, DJ761 and DJ694, were chosen for transcript quantification. Prior to gene quantification, reverse transcription, an essential step in the experimental procedure, was assessed for the error it incorporated into quantification. Specifically, an exogenous molecule was used to ensure that unsuccessful reverse transcription reactions had the potential to be identified and, soon after, discarded. This was achieved through the use of a spike RNA molecule, Luciferase. Luciferase was shown to be a diagnostic tool that can be used in determining reverse transcription efficiency. Eight genes were chosen from the aforementioned DJ lines and quantitative PCR revealed that the natural regulation of some genes were comparable to the, previously obtained, expression pattern of the enhancer trap line. Although the expression of other genes did not correlate to that of the enhancer trap lines, all genes exhibited expression patterns that were age-associated. The known functions of these candidate genes and the relevant homologues are discussed. These findings validate the use of the enhancer trap technique in the identification of candidate genes involved in the aging process.
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