Browsing SUNY at Fredonia by Subject "Gene targeting."
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Evidence for IRES Mediated Translation of Gurken.The gene gurken (grk) in Drosophila melanogaster is required to establish the anterior/posterior and dorsal/ventral axes of the egg. When insulin levels are high such as when food is readily available, grk is translated using a common mechanism that requires recognition of the 7-methylguanosine cap at the 5’ end of the RNA. Translation of grk requires Vasa activity which is inhibited through phosphorylation is spindle-B (spn-B) mutants. It was discovered in our lab that Insulin/Insulin-like Signaling (IIS) mutations or inhibition of TOR by rapamycin result in increased grk translation in spn-B mutants thereby overcoming this cap-dependent block. Based on these data, we hypothesize that the grk 5’ UTR contains an Internal Ribosomal Entry Site (IRES). An IRES provides an alternative mechanism of translation that occurs through use of a secondary structure in the 5’ UTR in the mRNA. The utility of the IRES is that it allows for translation of important mRNA’s even in the absence of canonical cap-dependent translation factors. These conditions can arise in nature when insulin levels or nutrient availability is low. The presence of an IRES will be explored in vivo through a comparison of transgenic reporter lines containing different luciferase reporter constructs. These constructs will be used to test the hypothesis that the grk 5’ UTR contains an IRES and examine the effect of nutrient limitation, inhibition of TOR, or IIS mutations on grk translation. To localize the IRES within the GRK 5' UTR selective deletion mutations will be made in secondary structures identified through selective 2’-Hydroxyl Acylation and Primer Extension (SHAPE) analysis. The activity of these reporter lines will be analyzed through assaying these transgenic lines. This will allow for the identification of the presence and location of the IRES within the grk 5’ UTR. Here we present data demonstrating the activity of reporter constructs that have been generated by fusion with the 5' UTR of grk. Additionally we propose a new technique using GRNA chromatography to identify the presence of an IRES as well as important secondary structures needed for IRES function using the same reporter constructs.