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dc.contributor.authorWener, Kristinaeng
dc.contributor.authorRestrepo, Ricardoeng
dc.contributor.corporatenameUniversity of Missouri-Columbia. Office of Undergraduate Researcheng
dc.contributor.meetingnameSummer Undergraduate Research and Creative Achievements Forum (2006 : University of Missouri--Columbia)eng
dc.date.issued2006eng
dc.descriptionAbstract only availableeng
dc.descriptionFaculty Mentor: Dr. Steven Notwehr, Biological Scienceseng
dc.description.abstractThe trans-Golgi network (TGN) reporter protein A-ALP, which is composed of the luminal (LD) and transmembrane domain (TMD) of TGN resident protein ALP fused to the cytosolic domain (CD) of Ste13, allows us to detect its arrival to the vacuole and therefore its rate of processing in S. cerevisiae. A-ALP is useful in gene screenings, being cleaved at a C-terminal site by a pep4 dependent carboxypeptidase in the vacuole, enabling detection for an assortment of mutations in the CD region. Alternatively, the Cps1 gene codes for the vacuolar enzyme, carboxypeptidase yscS; this cleaves the substrate CBZ-Gly-Leu. This mechanism allows for the leucine auxitrophic Cps1 strain to grow without the presence of any other leucine source, making it a good strategy for a gene selection, allowing identification for specific mutations. A Ste13(CD +TMD)-Cps1(LD) reporter construct was made to see if it would behave in the same manner and remains inactive until reaching the vacuole. For comparison, four derivatives of this strain were made with mutations in the Ste13 region with known processing rates when using A-ALP, verified by DNA sequencing. Western blotting was used to assess the expression and steady state processing of each protein. To ensure the proteins could not be processed prior to reaching the vacuole, the strains were grown up on plates with the CBZ-Gly-Leu substrate. An imunoprecipitation experiment will now be done to show processing by using radioisotope markers and to determine a half-time to determine the functional ability of each Ste13-Cps1 construct.eng
dc.identifier.urihttp://hdl.handle.net/10355/921eng
dc.publisherUniversity of Missouri--Columbia. Office of Undergraduate Researcheng
dc.relation.ispartofcommunityUniversity of Missouri-Columbia. Office of Undergraduate Research. Undergraduate Research and Creative Achievements Forumeng
dc.source.urihttp://undergradresearch.missouri.edu/forums-conferences/abstracts/abstract-detail.php?abstractid=755eng
dc.subjecttrans-golgi networkeng
dc.subjectgene selectioneng
dc.subjectbiochemistryeng
dc.titleCreation of a gene selection method using a Ste13-Cps1 construct in S. cerevisiae [abstract]eng
dc.typeAbstracteng


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