Ispitivanje proteinskih interakcija u kvascu Saccharomyces cerevisiae koristeći metodu in vivo koimunoprecipitacije i niz endogena obilježenih otvorenih okvira čitanja

master's thesis

• Title (english): Testing for protein interactions in yeast Saccharomyces cerevisiae using the targeted in vivo co-immunoprecipitation assays and the array of endogenously tagged open reading frames
• Author: Lucija Beneti
• Mentor: Zoran Zgaga (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biology and Microbial Genetics); Zagreb
• Defense date and country: 2006-04-06, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Razvijen je novi sistem za detekciju proteinskih interakcija u kvascu. Formirana je zbirka sojeva kvasaca koji eksprimiraju proteine, C-terminalno obilježene različitim epitopima sa svojih endogenih promotora, uključene u DNA replikaciju, rekombinaciju i popravak. „Mamac“ protein sadrži V5 i 6xHis, a “plijen” se razlikuje od “mamca” u 3xVSV umjesto 6xHis. U kvaščevoj stanici protein obilježen sa His će formirati fiziološki kompleks sa svojim interakcijskim partnerima pa koristeći koimunoprecipitacijsku metodu možemo izolirati „mamac“ i proteine koji s njim asociraju. Sistem je najprije korišen za reprodukciju interakcija opisanih u literaturi, a zatim i za detekciju proteina koji stupaju u interakcije sa Sae2, proteinom za kojeg se prepostavlja da ima ulogu u popravku dvostrukih lomova u DNA. Testiranje je rezultiralo pozitivnom interakcijom Sae2 sa Mre11, katalitičkom podjedinicom MRX kompleksa koji ima značajnu ulogu u procesiranju dvostrukih lomova u toku mejoze i mitoze.

Abstract (english)

Novel system for the detection of protein-protein interactions has been developed. A yeast strain collection was generated expressing full-length proteins involved in DNA replication, repair and recombination, C-terminally tagged from their endogenous promoters with exogenous protein tags, i.e the bait with a 6-His tag while the preys are fused to a 3xVSV epitope tag. In the yeast cell, bait protein is going to form a physiological complex with its associating partners so using pull down assays bait and bait-associated proteins could be isolated and detected. System was first used to reproduce the previously described interactions from the literature and than to detect proteins interacting with Sae2, a protein with a proposed role in the repair of double-strand breaks (DSB). This small-scale screen resulted in positive interaction with Mre11, the catalytic subunit of the MRX complex that plays an important role in processing DSBs during meiosis and mitosis.

• Keywords: endogeno obilježeni proteini; Mre11; Sae2 interakcijski partneri
• Keywords (english): endogenously tagged proteins; Mre11; Sae2-interacting partners
• Language: croatian
• URN:NBN: urn:nbn:hr:159:964683
• Study programme: Title: Biotechnology; specializations in: Biochemical-Microbiology; Course: Biochemical-Microbiology; Study programme type: university; Study level: graduate; Academic / professional title: diplomirani inženjer biotehnologije; smjer biokemijsko-mikrobiološki (diplomirani inženjer biotehnologije; smjer biokemijsko-mikrobiološki)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-09-11 08:57:09