The Xenopus laevis oocyte was examined as a model system for investigating insulin receptor function. The role of extracellular calcium on insulin-stimulated deoxyglucose uptake (ISDU) in the 𝘟𝘦𝘯𝘰𝘱𝘶𝘴 𝘭𝘢𝘦𝘷𝘪𝘴 oocyte was investigated. It was determined that removal of calcium from the medium did not alter the rate of ⁴⁵Ca²⁺ release from oocytes preloaded with ⁴⁵CaCl₂ In contrast to earlier reports using tissue explants and cultured cells, the insulin response in oocytes is not sensitive to a range of extracellular calcium concentrations from 1 μM to 10 mM. However, treatment of oocytes with 1 mM EGTA, in the absence of Ca²⁺, prior to, during or within 5 minutes of insulin addition resulted in a 2-4 fold inhibition of ISDU. To further investigate the event(s) in insulin signalling inhibited by EGTA, the number of receptors for insulin on the oocyte must be increased. To this end we have investigated the effects of the 5' and 3' untranslated regions as well as the coding region of mRNA on translational efficiency in reticulocyte lysate and oocytes. The results obtained in Xenopus oocytes are consistent with earlier cell-free data (Falcone and Andrews, 1991). We have demonstrated that replacing the cognate 5' UTR with the Xenopus beta globin 5' UTR appropriately linked to a consensus sequence for efficient translation initiation (ACCATGG) results in increased in translation in Xenopus oocytes. In vitro synthesized preprolactin transcript injected into oocytes was found to be functionally stable for several days (D. Andrews unpublished data). Stabilization of the preprolactin 3' transcript was localized to the UTR. Furthermore, inserting the preprolactin 3' UTR downstream of another coding region resulted in stabilization of the modified transcript. These results provided a basis for improving expression of cloned human insulin receptor in Xenopus oocytes. By optimizing the 5' and 3' UTR's of the insulin receptor clone we were successful in expressing high levels of insulin receptors in Xenopus oocytes. Effects of the coding region on translation were also investigated and we provide evidence that sequences in the coding region modulate translational efficiency. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22775 |
| Date | January 1993 |
| Creators | Vassilakos, Aikaterini |
| Contributors | Andrews, D. W., Ananthanarayanan, V. S., Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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