Upotreba PUB puls tehnike u istraživanju visokoorganizirane strukutre kromatina u humanim stanicama

master's thesis

• Title (english): Use of PUB pulse approach to study high order chromatin structure in human cells
• Author: Anamarija Jurišić
• Mentor: Višnja Gaurina Srček (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Cell Technology, Application and Biotransformations); Zagreb
• Defense date and country: 2013-09-19, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Pravilna organizacija genoma je osnova stanicama za ispravnu funkciju genoma. U ovom radu testirana je nova in vivo PUB (Proximity Utilizing Biotinylation) metodologija za proučavanje kromatina u blizini proteina od interesa Metoda se temelji na ko-ekspresiji proteina od interesa fuzioniranog bakterijskom biotin ligazom (BirA) zajedno sa histonom fuzioniranim biotin akceptor peptidom (BAP) koji je specifično biotiniliran od strane BirA. Ugradnja BAP histona u kromatin koji se nalazi u blizini BirA fuzionirane s proteinom od interesa rezultira biotinilacijom BAP domene vezane na histon koja se može detektirati metodom Western blotom i Imunofluorescencijom. Kao eksperimentalni model za testiranje ove tehnike odabrani su protein Emerin i histoni H2A i H3.1. Ovaj pristup pruža vrijedne informacije o stanju kromatina u blizini određenog proteina od interesa, što može pomoći u određivanju epigenetičkog statusa stanica na razini intranuklearne lokalizacije.

Abstract (english)

Proper organization of the genome is essential for faithful genome function and in this work a new methodology PUB (Proximity Utilizing Biotinylation) was tested to study chromatin in proximity of the protein of interest in vivo. Method is based on co-expression of the protein of interest, fused with the bacterial biotin ligase BirA together with a histone fused with BAP (biotin acceptor peptide), which is specifically biotinylated by BirA. With the incorporation of BAP histones into chromatin, the chromatin located in proximity of the BirA-fusion of protein of interest is preferentially biotinylated and it can be detected by Western blot, Immunoflourescence ect. Protein Emerin and histones H2A and H3.1 were chosen as an experimental model to validate PUB method. This approach could provide valuable information about the state fo chromatin around the particular protein of interest and it could help in determinating the epigenetic status of the cells on the level of intra-nuclera localization.

• Keywords: genom; kromatin; PUB; BirA; BAP; intranuklearna lokalizacija
• Keywords (english): genome; chromatin; PUB; BirA; BAP; intranuclear localization
• Language: croatian
• URN:NBN: urn:nbn:hr:159:490734
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-05-04 12:06:59