Potrditev novih interaktorjev variant proteina Pig1 kvasovke Saccharomyces cerevisiae

Žganjar, Mia (author), Petrovič, Uroš (mentor)

Za naravne populacije so značilne kompleksne ravni uravnavanja celičnih procesov, ki so običajno pod posrednim ali neposrednim vplivom okolja. Raznolikost med osebki populacije je zato moč pripisati raznolikemu medsebojnemu povezovanju teh ravni, izražanje lastnosti pa pripisujemo pripadajočim razlikam genetskih ozadij. Za odkrivanje genske arhitekture in molekulskih mehanizmov kompleksnih lastnosti uporabljamo številne pristope povezovanja fenotipa z genotipom. Vzročno povezovanje genotipa in fenotipa med drugim omogoča analiza določanja lokusov kvantitativnih lastnosti (ang. Quantitative Trait Locus analysis; QTL) ter njene izpeljanke. Z analizo QTL je bila prepoznana tudi kompleksna lastnost sposobnosti kopičenja založnih lipidov v kvasovki S. cerevisiae [1]. Predlagani so bili kandidatni vzročni geni, od katerih PIG1 predstavlja gen z najvišjo napovedno močjo fenotipa na osnovi variante lokusa kvantitativne lastnosti. Alelni varianti PIG1 preučevanih sevov BY4741 in AWRI1631 se razlikujeta v številnih polimorfizmih, ki predstavljajo več kot 2 % zaporedja gena. Raznolikost variant zato dopušča spremenjen interaktom in divergentno funkcionalnost. Ob preiskovanju vloge PIG1 v lipidnem metabolizmu smo uporabili dopolnjevanje proteinskih in genetskih interakcijskih profilov z določanjem novih genetskih in proteinskih interakcijskih partnerjev variant PIG1. Tako smo želeli preučiti pomen perturbacije interaktoma, odvisnega od genetskega ozadja seva. V sklopu magistrskega dela smo, za določevanje genetskih interakcij, izvedli test celične rasti ob prisotnosti rapamicina. Določili smo dve novi genetski interakciji gena PIG1 in sicer negativno genetsko interakcijo z RML2 ter pozitivno genetsko interakcijo s PHO23. Ugotovili smo, da kombinacija alelov seva AWRI1631v genetskem ozadju seva BY4741 povzroča hibridno nekompatibilnost s še neznanim genetskim dejavnikom. Razlike v interaktomih med sevoma smo podkrepili z visokozmogljivo različico metode dvohibridnega sistema kvasovke. Ta pristop nam je omogočal določiti nove kandidatne proteinske interaktorje variant proteina Pig1. V nadaljevanju smo s klasičnimi pristopi potrdili zgolj že znano interakcijo s proteinom Gsy2.

• Language: Slovenian
• Keywords: genetske interakcije, proteinske interakcije, lokus kvantitativne lastnosti, PIG1
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2021
• PID: 20.500.12556/RUL-129721
• COBISS.SI-ID: 79701251
• Publication date in RUL: 07.09.2021

Secondary language

• Language: English
• Title: Confirmation of novel yeast Saccharomyces cerevisiae Pig1 protein variant interactors

Abstract

Natural populations harbour complex levels of cellular regulation, which are usually directly or indirectly influenced by environmental factors. The variance of a trait in a population can therefore be explained by its underlying diverse interconnection of those regulation levels, which are based on the corresponding genetic background. In an aim of discovering the genetic architecture and molecular mechanisms of complex traits, we use several approaches to associate phenotype with genotype. One way of casual linking the genotype to phenotype is in using the quantitative trait locus (QTL) analysis and its extensions. Using such an analysis, accumulation of lipid storage in the common yeast S. cerevisiae has been determined as a quantitative, complex trait. Among proposed causative candidate genes, PIG1 had the highest QTL prediction power of the phenotype based on different loci of quantitative traits [1]. Its allelic polymorphic variance between the commonly used laboratory BY4741 strain and the AWRI1631 wine strain is higher than 2% and allows for altered interactome and its consequential divergent functionality. In the scope of investigating the role of PIG1 in lipid metabolism, we aimed to complement its interaction profiles by determining new genetic and protein interaction partners, based on the allelic variant of the PIG1 gene. Thus, we wanted to analyse the importance of perturbation of the interactome, which depends on the underlying genetic background. To determine genetic interactions, we performed a cell growth test in the presence of rapamycin. Two new interactions of the PIG1 were identified, namely a negative interaction with RML2 and a positive interaction with PHO23. We found that the combination of the AWRI1631 strain alleles in the BY4741 genetic background causes hybrid incompatibility with a yet unknown genetic factor. The interactome divergence between studied stains was supported by a high-throughput variant of the yeast two-hybrid method. With this so-called barcode-fused yeast two-hybrid method we were able to identify new candidate protein interactors of Pig1 variants. With the follow-up classical approaches, we were only able to confirm the previously known protein interaction with Gsy2.

• Keywords: Genetic interactions, protein interactions, quantitative trait locus, PIG1