Gpr1 mutagenesis libraries reveal novel mutations altering glucose signaling
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Typ
Examensarbete för masterexamen
Master Thesis
Master Thesis
Program
Biotechnology (MPBIO), MSc
Publicerad
2017
Författare
Lawenius, Lina
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
Glucose sensing in Saccharomyces cerevisiae is mediated by indirect or direct glucose sensing mechanisms. One direct mechanism is the GPCR system constituted by the Gpr1 receptor and its cognate G protein Gpa2. In response to glucose cAMP is generated which in turn binds to the regulatory subunit of PKA causing the catalytic subunits to dissociate and phosphorylate downstream targets. A parallel pathway constituted by Ras2 can also activate the same pathway. However, the downstream targets of both pathways are poorly characterized. This work aimed to generate a range of mutated Gpr1 receptors through development of a reporter assay utilizing the SUC2 promoter which is repressed in high extracellular glucose concentrations and de-repressed when glucose becomes limiting, (below 5mM). The repression and de-repression mechanisms are regulated by factors binding the two activation sites of the SUC2 promoter (termed SUC2A and SUC2B site). The activation sites were exploited and a set of SUC2 promoters were engineered to drive the expression of a reporter construct in response to glucose. The same reporter constructs were used to screen a mutagenesis library for Gpr1 receptors with altered glucose activations profiles. In conclusion, we found that: 1) The SUC2 pathway can be used as readout of the Gpr1 receptor. 2) Deletion of the SUC2A activation site provides an altered activation phenotype for PSUC2 and can be utilized for screening a Gpr1 mutagenesis library. 3) Gpr1 mutants with altered glucose activation profiles were generated with both higher basal activity and increased response to glucose. These receptors are hypothesized to be valuable tools in generating synthetic glucose bio-sensors.
Beskrivning
Ämne/nyckelord
Livsvetenskaper , Biokemi och molekylärbiologi , Cellbiologi , Cell- och molekylärbiologi , Life Science , Biochemistry and Molecular Biology , Cell Biology , Cell and Molecular Biology