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Construction of Leo1 Mutants to understand their role in Intellectual Disability
University of Skövde, School of Bioscience.
2020 (English)Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Intellectual Disability is characterized by a substantially low level of mental abilities in daily life. There are three mutations observed in Leo1 conserved regions in patients presenting an intellectual disability. Leo1 protein is part of the Paf1 complex. This complex has several functions in different molecular and cellular processes. The Paf1 complex has four conserved subunits in eukaryotes including Leo1, Paf1, Ctr9, and Cdc73. It is already established that Leo1 mutants have a role in chromatin regulation. This project is about the construction of Leo1 mutants to study the Leo1 mutations affecting the function of the Paf1 complex on chromatin regulation in fission yeast. For this project, the yeast model Schizosaccharomyces pombe will be used because of its genetic malleability and the conservation of molecular processes between yeast and human. The project is based on homologous recombination in fission yeast using two strategies to create the mutants. Both strategies involve the disruption of the endogenous Leo1 gene with the selection marker and then the replacement of the disrupted Leo1 alleles with Leo1 alleles carrying the intended mutations. The PCR based site-directed mutagenesis was used to construct 3 Leo1 mutant alleles during the first stage of the project. There are two types of primers utilized; one with intended mutations and second to amplify sequence. The PCR products are transformed into yeast using the electroporation method. The project is successful in the synthesis of PCR products for constructing the Leo1 mutant alleles and the clone’s validation assay is also developed.

Place, publisher, year, edition, pages
2020. , p. 34
National Category
Medical Bioscience
Identifiers
URN: urn:nbn:se:his:diva-19220OAI: oai:DiVA.org:his-19220DiVA, id: diva2:1495012
External cooperation
Karolinska Institute
Subject / course
Bioscience
Educational program
Molecular Biotechnology - Master’s Programme, 60 ECTS
Supervisors
Examiners
Available from: 2020-11-04 Created: 2020-11-04 Last updated: 2020-11-04Bibliographically approved

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